Rna purification methods

ABSTRACT

Methods for purifying RNA from a sample, comprising one or more steps of tangential flow filtration, hydroxyapatite chromatography, core bead flow-through chromatography, or any combinations thereof. These techniques are useful individually, but show very high efficiency when used in combination, or when performed in particular orders. The methods can purify RNA in a highly efficient manner without unduly compromising potency or stability, to provide compositions in which RNA is substantially cleared of contaminants. Moreover, they can be performed without the need for organic solvents.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/799,705, filed Mar. 15, 2013, the complete contents of which arehereby incorporated herein by reference for all purposes.

TECHNICAL FIELD

This invention is in the field of RNA purification, and in particularmethods for the large-scale purification and formulation of large RNAfrom complex samples such as samples obtained after in vitrotranscription of RNA, for pharmaceutical use, for example for use inimmunising animals.

BACKGROUND ART

RNA is emerging as an innovative candidate for a variety ofpharmaceutical applications, but efficient purification is continuing tobe a challenge. This is partly due to the different types andcombinations of undesired contaminants in a sample that need to beseparated from a desired RNA species to obtain a pure RNA sample. Suchcontaminants are typically components and by-products of any upstreamprocesses, for example RNA manufacture. Where in vitro transcription isused to manufacture large RNA, following successful transcription thesample typically contains the desired RNA species alongside variouscontaminants such as undesired RNA species, proteins, DNA or fragmentsthereof, pyrophosphates and free nucleotides.

Commercial downstream applications (e.g. formulation and use as apharmaceutical composition and/or vaccine) pose further constrains onany purification method for large RNA requiring (i) a high degree ofpurity while retaining RNA stability and functionality; (ii)compatibility with any formulation requirements of the RNA for in vivodelivery; and (iii) compliance with good manufacturing practices.Furthermore, in order to facilitate industrial applications, any RNApurification method must enable consistent, cost- and time-efficientoperation (e.g. quick, easy, reproducible, high yield purification on alarge scale).

Methods for the purification of large RNA are known in the art. Pascoloet al. (2006) describes a method for the purification of mRNA from an invitro transcription reaction sample in analytical scale (purification of25 μg RNA in 20 μl sample volume). The method involves DNase treatmentfollowed by precipitation of the longer mRNA with lithium chloride.However, the authors report that this method does not provide RNA ofhigh purity, as it does not completely remove contaminants such as DNAand protein. Furthermore, the method involves the use of organicsolvents and is laborious and time-consuming, involving as many as 36steps requiring extensive manual sample handling at differentconditions, including at least one overnight incubation step. Therefore,while this procedure may satisfy requirements for research andlaboratory-scale RNA purification, it suffers from a low degree of RNApurity, reproducibility and is unsuitable for purification ofpharmaceutical-grade RNA on a commercial scale for implementation in anindustrial process.

WO2008/077592 discloses a method for purifying large RNA on apreparative scale with ion-pairing reverse phase HPLC using a porousreversed stationary phase. It is reported that a particular advantage ofusing the specified porous stationary phase is that excessively highpressures can be avoided, facilitating a preparative purification ofRNA. However, the method involves the use of harsh organic solvents(e.g. acetonitrile) and high temperatures (78° C.) for the separationcolumn, and a low temperature (12° C.) for the sampler. The nature ofthe contaminant(s) that can be successfully separated from a desired RNAusing the method is not exemplified, including any requirements forpreceding steps such as DNase treatment Additionally, chromatographicseparation of RNA based on ion-pairing reverse phase HPLC or ionexchange resin are based on the molecule's total charge and may beeffective for purification of RNA molecules of up to about 4,000-5,000bases. However, the purification of larger RNA molecules suffers fromsize exclusion effects and poor recovery. Furthermore, it relies onelution of RNA using organic solvents, but these should ideally beavoided due to potential safety concerns about residues, high purchasecosts, their environmental impact, and potential detrimental effects onRNA stability and potency.

Thus there remains a need for further and improved RNA purificationmethods, and in particular for those that allow cost- and time-efficientpurification of large RNAs at an industrial scale with high yield andpharmaceutical-grade purity while retaining the stability, biologicalpotency and functionality of the RNA. There is a particular need forsuch methods where the starting sample is a complex biological samplesuch as those obtained after in vitro transcription of large RNA.

DISCLOSURE OF THE INVENTION

To address these needs, the invention provides a method for purifyingRNA from a sample, comprising one or more steps of tangential flowfiltration, hydroxyapatite chromatography, core bead flow-throughchromatography, or any combinations thereof. These techniques are usefulindividually, but show very high efficiency when used in combination, orwhen performed in particular orders. The methods can purify RNA in ahighly efficient manner without unduly compromising potency orstability, to provide compositions in which RNA is substantially clearedof contaminants. Moreover, they can be performed without the need fororganic solvents, and it is preferred that methods of the invention takeplace in aqueous conditions. A further advantage of the invention isthat uses components which are essentially disposable, meaning that theycan be prepared in thoroughly-cleaned form (in particular, RNase-freeform), used only once, and then discarded, so that carry-throughrun-to-run contamination can be avoided, which is particularly usefulwhen avoiding RNase contamination. The methods are also very quick.

In one embodiment, the invention provides a method for purifying RNAfrom a sample, wherein the method comprises one or more steps oftangential flow filtration.

In another embodiment, the invention provides a method for purifying RNAfrom a sample, wherein the method comprises one or more steps ofhydroxyapatite chromatography.

In another embodiment, the invention provides a method for purifying RNAfrom a sample, wherein the method comprises one or more steps of corebead flow-through chromatography.

In a useful embodiment, the method comprises a step of tangential flowfiltration and a step of hydroxyapatite chromatography. The step oftangential flow filtration preferably precedes the step ofhydroxyapatite chromatography.

In another useful embodiment, the method comprises a step of core beadflow-through chromatography and a step of hydroxyapatite chromatography.The step of core bead flow-through chromatography ideally precedes thestep of hydroxyapatite chromatography.

In any of the foregoing embodiments, the recited steps for RNApurification may be followed by one or more steps of buffer exchangee.g. comprising tangential flow filtration.

Thus the invention provides a method for the purification andformulation of RNA from a sample, wherein the method comprises one ormore steps of RNA purification and one or more steps of buffer exchange.Preferably, the at least one step of buffer exchange comprisestangential flow filtration.

In one embodiment, the invention provides a method for the purificationand formulation of RNA comprising two separate steps of tangential flowfiltration. Preferably, a first buffer is used in a first step oftangential flow filtration and a second different buffer is used in asecond step of tangential flow filtration. The first buffer and thesecond buffer are usually based on two different buffer salts. Forexample, the first buffer may be a Tris-based buffer whereas the secondbuffer may be a citrate buffer. Preferably, the first buffer is apurification buffer and the second buffer is a formulation buffer. Morepreferably, the purification buffer comprises a salt at a concentrationof between 50-500 mM e.g. 250 mM.

For example, the purification buffer may comprise a salt at aconcentration of between 0-500 mM, such as about 10 mM, about 20 mM,about 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM,about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM,about 500 mM, from about 10 mM to about 500 mM, from about 10 mM toabout 400 mM, from about 10 mM to about 300 mM, from about 10 mM toabout 250 mM, from about 20 mM to about 500 mM, from about 20 mM toabout 400 mM, from about 20 mM to about 300 mM, from about 20 mM toabout 250 mM, from about 30 mM to about 500 mM, from about 30 mM toabout 400 mM, from about 30 mM to about 300 mM, from about 30 mM toabout 250 mM, from about 40 mM to about 500 mM, from about 40 mM toabout 400 mM, from about 40 mM to about 300 mM, from about 40 mM toabout 250 mM, from about 50 mM to about 500 mM, from about 50 mM toabout 400 mM, from about 40 mM to about 300 mM, or from about 50 mM toabout 250 mM, etc.

In another embodiment, the invention provides a method for thepurification and formulation of RNA comprising a step of core beadflow-through chromatography, followed by a step of tangential flowfiltration. In a preferred embodiment, a first buffer is used in thestep of core bead flow-through chromatography and a second differentbuffer is used in the step of tangential flow filtration. Preferably,the first buffer is a purification buffer and the second buffer is adifferent formulation buffer. More preferably, the purification buffercomprises a salt, such as potassium chloride or sodium chloride. Mostpreferably, the purification buffer comprises potassium chloride at aconcentration of between 100-500 mM e.g. 250 mM.

For example, the purification buffer may comprise potassium chloride ata concentration of between 0-500 mM, such as about 50 mM, about 75 mM,about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM,about 350 mM, about 400 mM, about 450 mM, about 500 mM, from about 50 mMto about 500 mM, from about 50 mM to about 400 mM, from about 50 mM toabout 300 mM, from about 50 mM to about 250 mM, from about 75 mM toabout 500 mM, from about 75 mM to about 400 mM, from about 75 mM toabout 300 mM, from about 75 mM to about 250 mM, from about 100 mM toabout 500 mM, from about 100 mM to about 400 mM, from about 100 mM toabout 300 mM, or from about 100 mM to about 250 mM, etc.

In another embodiment, the invention provides a method for thepurification and formulation of RNA comprising a first step oftangential flow filtration, then a second step of hydroxyapatitechromatography, then a third step of tangential flow filtration. In apreferred embodiment, a first buffer is used in the first step oftangential flow filtration, a second different buffer is used in thesecond step of hydroxyapatite chromatography and a third differentbuffer is used in the third step of tangential flow filtration.Preferably, the first and second buffers are purification buffers andthe third buffer is a different formulation buffer. Preferably, thefirst buffer is free from sodium chloride and/or potassium chloride.Most preferably, in an additional step, sodium chloride and/or potassiumchloride is added to the RNA-containing sample at a final concentrationof between 100-500 mM, e.g. 250 mM or 500 mM, after the first step oftangential flow filtration and before the second step of hydroxyapatitechromatography.

For example, sodium chloride and/or potassium chloride may be added tothe RNA-containing sample at a final concentration of between 0-500 mM,such as about 50 mM, about 75 mM, about 100 mM, about 150 mM, about 200mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450mM, about 500 mM, from about 50 mM to about 500 mM, from about 50 mM toabout 400 mM, from about 50 mM to about 300 mM, from about 50 mM toabout 250 mM, from about 75 mM to about 500 mM, from about 75 mM toabout 400 mM, from about 75 mM to about 300 mM, from about 75 mM toabout 250 mM, from about 100 mM to about 500 mM, from about 100 mM toabout 400 mM, from about 100 mM to about 300 mM, or from about 100 mM toabout 250 mM, etc.

In another embodiment, the invention provides a method for thepurification and formulation of RNA comprising a first step of core beadflow-through chromatography, then a second step of hydroxyapatitechromatography, then a third step of tangential flow filtration. In apreferred embodiment, a first buffer is used in the first step of corebead flow-through chromatography, a second different buffer is used inthe second step of hydroxyapatite chromatography and a third differentbuffer is used in the third step of tangential flow filtration.Preferably, the first and second buffer is a purification buffer and thethird buffer is a different formulation buffer. Preferably, the firstbuffer is free from sodium chloride and/or potassium chloride. Mostpreferably, in an additional step, sodium chloride and/or potassiumchloride is added to the RNA-containing sample at a final concentrationof between 100-500 mM, e.g. 250 mM or 500 mM, after the first step ofcore bead flow-through chromatography and before the second step ofhydroxyapatite chromatography.

For example, sodium chloride and/or potassium chloride may be added tothe RNA-containing sample at a final concentration of between 0-500 mM,such as about 50 mM, about 75 mM, about 100 mM, about 150 mM, about 200mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450mM, about 500 mM, from about 50 mM to about 500 mM, from about 50 mM toabout 400 mM, from about 50 mM to about 300 mM, from about 50 mM toabout 250 mM, from about 75 mM to about 500 mM, from about 75 mM toabout 400 mM, from about 75 mM to about 300 mM, from about 75 mM toabout 250 mM, from about 100 mM to about 500 mM, from about 100 mM toabout 400 mM, from about 100 mM to about 300 mM, or from about 100 mM toabout 250 mM, etc.

The invention also provides a method for purifying RNA from a sample(such as the product of an in vitro transcription reaction), wherein theRNA is purified to at least 99% purity (e.g. ≧99.5%, ≧99.9%, or even≧99.95%) in less than 12 hours (e.g. <8 hours, <6 hours, <4 hours, or <2hours).

In the methods of the invention, steps will generally involve discardingmaterials which do not contain RNA (or which do not contain the desiredRNA species) while maintaining materials which contain RNA (or thedesired RNA species). Thus, where a technique splits a starting materialinto fractions, desired fractions will be retained while undesiredfractions can be discarded; similarly, if a technique retains undesiredmaterials but lets desired RNA flow through, the flow through will beretained.

The RNA

According to the invention, a desired RNA is purified from anRNA-containing sample. The desired RNA of the invention can bedouble-stranded but is preferably single-stranded. Where the RNA issingle-stranded, such as mRNA or a self-replicating RNA replicon, ittypically encodes one or more proteins, and at least one of these isusefully an immunogen as discussed below but can also be anynon-immunogenic therapeutic or prophylactic protein of interest (e.g. asa component of a gene therapy medicament). The desired RNA of theinvention can be circular, but is preferably linear.

The RNA can be (−)-stranded, but is preferably is (+)-stranded, suchthat it can be translated by cells without needing any interveningreplication steps such as reverse transcription. Preferred +-strandedRNAs are self-replicating, as described below. Preferably, the RNA isnot a natural viral RNA.

The RNA may be a small, medium, or large RNA. The number of nucleotidesper strand of a small RNA is from 10-30 (e.g. siRNAs). A medium RNAcontains between 30-2000 nucleotides per strand (e.g.non-self-replicating mRNAs). A large RNA contains at least 2,000nucleotides per strand e.g. at least 2,500, at least 3,000, at least4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000,at least 9,000, or at least 10,000 nucleotides per strand (e.g.self-replicating RNAs as described below). The molecular mass of asingle-stranded RNA molecule in g/mol (or Dalton) can be approximatedusing the formula: molecular mass=(number of RNA nucleotides)×340 g/mol.

As discussed in WO2011/005799, an RNA (particularly a self-replicatingRNA) can include, in addition to any 5′ cap structure, one or morenucleotides having a modified nucleobase. For instance, a RNA caninclude one or more modified pyrimidine nucleobases, such aspseudouridine and/or 5-methylcytosine residues. In some embodiments,however, the RNA includes no modified nucleobases, and may include nomodified nucleotides i.e. all of the nucleotides in the RNA are standardA, C, G and U ribonucleotides (except for any 5′ cap structure, whichmay include a 7′-methylguanosine). In other embodiments, the RNA mayinclude a 5′ cap comprising a 7′-methylguanosine, and the first 1, 2 or3 5′ ribonucleotides may be methylated at the 2′ position of the ribose.

A RNA used with the invention ideally includes only phosphodiesterlinkages between nucleosides, but in some embodiments it can containphosphoramidate, phosphorothioate, and/or methylphosphonate linkages.

The invention is particularly suitable for purifying self-replicatingRNAs. A self-replicating RNA molecule (replicon) can, when delivered toa vertebrate cell even without any proteins, lead to the production ofmultiple daughter RNAs by transcription from itself (via an antisensecopy which it generates from itself). A self-replicating RNA molecule isthus typically a +-strand molecule which can be directly translatedafter delivery to a cell, and this translation provides a RNA-dependentRNA polymerase which then produces both antisense and sense transcriptsfrom the delivered RNA. Thus the delivered RNA leads to the productionof multiple daughter RNAs. These daughter RNAs, as well as collinearsubgenomic transcripts, may be translated themselves to provide in situexpression of an encoded protein of interest (e.g. an immunogen), or maybe transcribed to provide further transcripts with the same sense as thedelivered RNA which are translated to provide in situ expression of aprotein (e.g. an immunogen). The overall results of this sequence oftranscriptions is a huge amplification in the number of the introducedreplicon RNAs and so the encoded immunogen becomes a major polypeptideproduct of the cells. Suitable self-replicating RNAs are disclosed inWO2012/006369 and WO2013/006838.

One suitable system for achieving self-replication is to use analphavirus-based RNA replicon. These +-stranded replicons are translatedafter delivery to a cell to give of a replicase (orreplicase-transcriptase). The replicase is translated as a polyproteinwhich auto-cleaves to provide a replication complex which createsgenomic −-strand copies of the +-strand delivered RNA. These −-strandtranscripts can themselves be transcribed to give further copies of the+-stranded parent RNA and also to give a subgenomic transcript whichencodes the immunogen. Translation of the subgenomic transcript thusleads to in situ expression of the immunogen by the infected cell.Suitable alphavirus replicons can use a replicase from a sindbis virus,a semliki forest virus, an eastern equine encephalitis virus, avenezuelan equine encephalitis virus, etc. Mutant or wild-type virusessequences can be used e.g. the attenuated TC83 mutant of VEEV has beenused in replicons (WO2005/113782).

A preferred self-replicating RNA molecule thus encodes (i) aRNA-dependent RNA polymerase which can transcribe RNA from theself-replicating RNA molecule and (ii) an immunogen. The polymerase canbe an alphavirus replicase e.g. comprising one or more of alphavirusproteins nsP1, nsP2, nsP3 and nsP4.

Whereas natural alphavirus genomes encode structural virion proteins inaddition to the non-structural replicase polyprotein, it is preferredthat a self-replicating RNA molecule of the invention does not encodealphavirus structural proteins. Thus a preferred self-replicating RNAcan lead to the production of genomic RNA copies of itself in a cell,but not to the production of RNA-containing virions. The inability toproduce these virions means that, unlike a wild-type alphavirus, theself-replicating RNA molecule cannot perpetuate itself in infectiousform. The alphavirus structural proteins which are necessary forperpetuation in wild-type viruses are absent from self-replicating RNAsof the invention and their place is taken by gene(s) encoding theimmunogen of interest, such that the subgenomic transcript encodes theimmunogen rather than the structural alphavirus virion proteins.

Thus a self-replicating RNA molecule useful with the invention may havetwo open reading frames. The first (5′) open reading frame encodes areplicase; the second (3′) open reading frame encodes an immunogen. Insome embodiments the RNA may have additional (e.g. downstream) openreading frames e.g. to encode further immunogens (see below) or toencode accessory polypeptides.

A self-replicating RNA molecule can have a 5′ sequence which iscompatible with the encoded replicase.

Self-replicating RNA molecules can have various lengths but they aretypically 5000-25000 nucleotides long e.g. 8000-15000 nucleotides, or9000-12000 nucleotides.

A RNA molecule useful with the invention may have a 5′ cap. This cap canenhance in vivo translation of the RNA. The cap can be a natural ornon-natural cap, and is generally attached to the RNA's 5′-terminalnucleotide by a 5′ to 5′ triphosphate linkage. Various cap structuresare known e.g. 7-methylguanosine (m7G), 3′-O-Me-m7G or “ARCA” (antireverse cap analog), m2,2,7G, unmethylated cap analogs, etc.

The 5′ nucleotide of a RNA molecule useful with the invention may have a5′ triphosphate group. In a capped RNA this may be linked to a7-methylguanosine via a 5′-to-5′ bridge. A 5′ triphosphate can enhanceRIG-I binding and thus promote adjuvant effects.

A RNA molecule may have a 3′ poly-A tail. It may also include a poly-Apolymerase recognition sequence (e.g. AAUAAA) near its 3′ end.

In one embodiment, a method of the invention is used to purify amodified mRNA; in another embodiment, a method of the invention is usedto purify an unmodified mRNA;

The RNA-Containing Sample

According to the invention, a desired RNA is purified from anRNA-containing sample. The composition of the sample will largely dependon the source of the RNA and any preceding purification steps. Themethods of the invention are particularly useful for purification of adesired RNA from in vitro transcription (IVT) sources. In theseembodiments, the sample contains a desired RNA species and typicallycontaminants, including non-desired RNA, DNA (e.g. template DNA fromIVT), proteins (e.g. RNA polymerase, capping enzyme, DNase, RNaseinhibitor), pyrophosphates and/or free nucleotides. Free nucleotides arefound in IVT mixtures either as un-reacted RNA precursors (e.g.ribonucleoside triphosphate) or as degradation products from DNAdigestion (e.g. deoxynucleoside monophosphate). A typical buffer for IVTreactions is a Tris-based buffer, for example 50 mM Tris pH 8.0. Aparticular advantage of using IVT is that a large excess of the desiredRNA species is produced in a controlled reaction and that modified basescan easily be introduced into the RNA.

Thus a method of the invention may include a pre-purification step ofRNA manufacture by IVT. Thus the invention provides a method forpreparing a purified RNA, comprising steps of: (i) performing in vitrotranscription to provide a sample comprising RNA; and (ii) purifying theRNA from the sample, comprising one or more steps of tangential flowfiltration, hydroxyapatite chromatography, core bead flow-throughchromatography, or any combinations thereof.

IVT produces RNA from a DNA template in a cell-free, controlledbiochemical reaction typically including enzymes (e.g. RNA polymerase,and usually capping enzymes), RNA precursors (e.g. ribonucleosidetriphosphate), DNA template, reducing agents (e.g. DTT), and a suitablebuffer. Following IVT the DNA template should be removed to avoid itspresence in the final product, and thus it can be digested (e.g. usingDNase) or removed. Where the RNA purification method includes tangentialflow filtration or core bead chromatography, it is preferred to removeDNA prior to purification, for example using DNase. In contrast, wherethe method uses hydroxyapatite chromatography the DNA can be removedwithout needing DNase treatment, although a step of DNase treatment canstill be used if desired.

However, the invention is not limited to RNA from in vitro transcriptionreactions and in some embodiments RNA is manufactured using in vivo(cell-based) transcription, chemical synthesis, or synthetic genomicsapproaches.

Methods of the invention are also useful for the purification of RNAfrom an RNA-containing sample where the sample is an RNA virus extract,an RNA-containing cell extract (e.g. derived from animal, plant orbacterial cells), or an RNA-containing environmental sample or extractsthereof.

Purification of RNA from an RNA-Containing Sample

RNA purification is used to remove impurities from compositionscomprising a particular RNA of interest. Different purification stepscan be used to isolate the RNA of interest from non-RNA components ofthe composition (e.g. DNA and proteins), as well as from othercontaminant RNA.

Methods of the invention use one or more of three techniques: tangentialflow filtration; hydroxyapatite chromatography; and/or core beadflow-through chromatography. These methods can all be performed underaqueous conditions, so methods of the invention do not require the useof organic solvents, and are ideally performed without the use oforganic solvents, in particular without the use of organic solvents thatmay be toxic when administered to humans as part of a pharmaceuticalcomposition and/or which may adversely impact on the stability of largeRNAs. Methods of the invention are therefore ideally performed withoutthe use of acetonitrile, chloroform, phenol and/or methanol. Ideally,they can be performed without using any organic solvents.

Methods of the invention can conveniently be performed at roomtemperature.

Tangential Flow Filtration (TFF)

According to the invention, tangential flow filtration (TFF) may be usedto purify a RNA of interest by removing lower molecular weight species.Thus a method of the invention can comprise one or more steps of TFF.TFF is particularly useful for the purification of large RNA species.The inventors have shown that high yield (at least 90-95%) and purity(at least 90-99.9%) can be achieved using TFF for RNA purification,while retaining the stability and potency of the purified RNA. Usefully,TFF also permits buffer exchange (dialysis) at the same time aspurification (or TFF can be used with purified RNA as a separate bufferexchange step e.g. to change to a final formulation buffer. TFF is easyto operate, time-efficient (only about 70 minutes for both RNApurification and buffer exchange) and prevents contamination (e.g. withRNAse) due to the ability to operate as a closed system. TFF isparticularly useful for the removal of free nucleotides from an IVTmixture.

TFF involves passing a liquid containing the sample tangentially acrossa filter membrane. Thus TFF contrasts with dead-end filtration, in whichsample is passed through a membrane rather than tangentially to it. InTFF the sample side is typically held at a positive pressure relative tothe filtrate side. As the liquid flows over the filter, componentstherein can pass through the membrane into the filtrate. Where an IVTreaction sample is used, ribonucleoside triphosphates, small nucleicacid fragments such as digested template DNA, and/or other undesiredcomponents are typically removed in the filtrate whereas long RNA isrecovered from the retentate. Many TFF systems are commerciallyavailable (e.g. using hollow fibres such as those available from GEHealthcare and Spectrum Labs). The molecular weight cut-off (MWCO) of aTFF membrane determines which solutes can pass through the membrane(i.e. into the filtrate) and which are retained (i.e. in the retentate).The MWCO of a TFF filter used with the invention will be selected suchthat substantially all of the solutes of interest (i.e. desired RNAspecies) remains in the retentate, whereas undesired components passinto the filtrate. The retentate may be re-circulated to the feedreservoir to be re-filtered in additional cycles. Compared to dead-endfiltration, the retentate is washed away during the filtration process,minimising the clogging of the membrane which is known in the art as“membrane fouling”, maintaining a high, steady filtration rate acrossthe membrane, and increasing the length of time the process can becontinuously operated.

Parameters for operating TFF according to the invention will be selectedsuch that impurities can permeate the filter membrane whereas the RNA ofinterest is retained, without significantly affecting RNA integrityand/or potency.

The average pore size of a filter membrane is referred to in the art as“membrane pore size”. Membrane pore size is usually stated in kDa andrefers to the average molecular mass of the smallest particle ormacromolecule the membrane is likely to retain. Alternatively, membranepore size can be stated in μm and refers to the diameter of the smallestparticle the membrane is likely to retain. The diameter is proportionalto the molecular mass for molecules of a similar shape (e.g. sphericalmolecules). For example, a membrane pore size of 500 kDa is equivalentto a membrane pore size of approximately 0.02 μm for a sphericalmolecule.

The inventors have found that a membrane pore size of between 250 and1000 kDa is useful when purifying large RNA e.g. between 250 and 750kDa, or preferably between 400 and 600 kDa. A membrane having a poresize of about 500 kDa is particularly preferred. Preferably, themembrane pore size is selected such that the ratio of the size of theRNA molecule of interest to the membrane pore size is at least 1.5:1(e.g. at least 2:1, at least 3:1, at least 4:1, at least 5:1 or at least6:1) and/or that the ratio of the size of the largest non-RNA impurityto the membrane pore size is at least 1:1.5 (e.g. at least 1:2, at least1:3, at least 1:4, at least 1:5 or at least 1:6).

Where a sample comprises a desired RNA and a non-desired RNA species ofa different size, the method may include two or more steps of tangentialflow filtration, wherein each step uses a different membrane pore sizesuch that in one step smaller molecules than the RNA of interest areremoved and the RNA-containing retentate fraction is retained, and inone or more additional steps larger molecules than the RNA of interestare removed and the RNA of interest is recovered from the filtrate. Suchmethods may be combined with hydroxyapatite chromatography and core beadflow-through chromatography as described below. Thus in one embodiment,the invention provides a method for purifying RNA from a sample, whereinthe method comprises a first step of tangential flow filtration using afirst membrane, optionally followed by (a) further step(s) of core beadflow-through chromatography and/or hydroxyapatite chromatography,followed by a second step of tangential flow filtration using a secondmembrane, wherein the first and second membranes have different poresizes such that the first membrane retains the RNA of interest in theretentate and the second membrane permits the passage of the RNA ofinterest through the pores of the membrane into the filtrate and retainsimpurities in the retentate.

TFF may be carried out using any suitable filter membrane. The inventorshave found that a hollow fibre filter is particularly advantageous. Ahollow fibre filter typically comprises a multitude (bundle) of hollow,open-ended tubes (fibres), through which the liquid containing thesample is passed from the feed side to the retentate side. The walls ofthe tubes are composed of a membrane (the filter membrane), whichtypically has a three-dimensional internal structure of interconnectedcavities (pores). Common filter membrane polymers that can be used inthis invention are polysulfone (PS), polyethersulfone (PES). PES may bemodified (mPES) to have increased hydrophilicity and to have higherpermeate flux rates than un-modified PES. Several different methods areknown to transform hydrophobic PES membranes into hydrophilic PESmembranes. The inventors have found that hydrophilic membranes andparticularly modified polyethersulfone (mPES) membranes are particularlyadvantageous for RNA purification.

TFF membranes may vary according to their effective surface area. Theeffective membrane surface area is typically stated in cm² and refers tothe total surface of the filter membrane that is exposed to the sample.The effective surface area for hollow fibre membranes depends on theaverage diameter and effective length of the fibres and the total numberof fibres. The inventors have found that the effective membrane area caninfluence the operation time of the TFF method and the efficiency of RNApurification and buffer exchange. Processing parameters determined forsmall-scale volumes can be used for larger volumes by maintaining theeffective length of the filter and increasing the effective membranearea (e.g. by increasing the average fibre diameter and/or the totalnumber of fibres).

A TFF method may vary according to the transmembrane pressure that isapplied during the process. Transmembrane pressure is the averagepressure differential between the feed side and the filtrate side of thefilter membrane. Ideally, the transmembrane pressure is chosen so that ahigh flux of the fluid across the membrane is achieved while maintainingefficient separation of the RNA of interest from any impurities andavoiding the formation of a gel layer on the surface of the filtermembrane. The inventors have found that a transmembrane pressure between1 psi (6895 Pa) and 5 psi (34475 Pa) is preferred. Ideally, thetransmembrane pressure is set to about 2 psi (13790 Pa).

A TFF method may vary according to the shear rate, or also known in theart as the retentate velocity. The shear rate is typically stated inreciprocal seconds (s⁻¹) and can be calculated according to formulaeknown in the art. Ideally, the shear rate is chosen so that a high fluxof the fluid through the filter is achieved while maintaining RNAintegrity and avoiding the formation of a gel layer on the surface ofthe filter membrane. The inventors have found that a shear rate betweenabout 500-5000 s⁻¹ is preferred. More preferably, a shear rate of about800 s⁻¹ is used.

For example, a shear rate of about 500 s⁻¹, about 600 s⁻¹, about 700s⁻¹, about 800 s⁻¹, about 900 s⁻¹, about 1000 s⁻¹, about 1100 s⁻¹, about1200 s⁻¹, about 1300 s⁻¹, about 1400 s⁻¹, about 1500 s⁻¹, about 1600s⁻¹, about 1700 s⁻¹, about 1800 s⁻¹, about 1900 s⁻¹, about 2000 s⁻¹,about 2500 s⁻¹, about 800 s⁻¹, about 3000 s⁻¹, about 3500 s⁻¹, about4000 s⁻¹, about 4500 s⁻¹, about 5000 s⁻¹, from about 500 s⁻¹ to about5000 s⁻¹, from about 500 s⁻¹ to about 4000 s⁻¹, from about 500 s⁻¹ toabout 3000 s⁻¹, from about 500 s⁻¹ to about 2000 s⁻¹, from about 500 s⁻¹to about 1000 s⁻¹, from about 600 s⁻¹ to about 5000 s⁻¹, from about 600s⁻¹ to about 4000 s⁻¹, from about 600 s⁻¹ to about 3000 s⁻¹, from about600 s⁻¹ to about 2000 s⁻¹, from about 600 s⁻¹ to about 1000 s⁻¹, fromabout 700 s⁻¹ to about 5000 s⁻¹, from about 700 s⁻¹ to about 4000 s⁻¹,from about 700 s⁻¹ to about 3000 s⁻¹, from about 700 s⁻¹ to about 2000s⁻¹, from about 700 s⁻¹ to about 1000 s⁻¹, from about 800 s⁻¹ to about5000 s⁻¹, from about 800 s⁻¹ to about 4000 s⁻¹, from about 800 s⁻¹ toabout 3000 s⁻¹, from about 800 s⁻¹ to about 2000 s⁻¹, or from about 800s⁻¹ to about 1000 s⁻¹, etc., may be used. A fluid may be fed into theTFF system in addition to the RNA-containing sample. The fluid istypically a buffer. The choice and composition of the buffer mayinfluence the efficiency of RNA purification and/or buffer exchange,levels of protein aggregation, RNA-protein separation and RNA stability.Typical buffer include those based on citric acid and Tris. Theinventors have found that a Tris based buffer, for example containing 10mM Tris, performs particularly well. Preferably, the buffer pH isbetween 6.5 and 9.0, between 7.0 and 8.5, between 7.5 and 8.5, between7.8 and 8.2. More preferably, the sample buffer pH is 8.0.

For example, the pH of the buffer may be about 6.5, about 7.0, about7.5, about 7.8, about 8.0, about 8.2, about 8.5, about 9.0, betweenabout 6.5 to about 9.0, between about 6.5 to about 8.5, between about6.5 to about 8.2, between about 6.5 to about 8.0, between about 7.0 toabout 9.0, between about 7.0 to about 8.5, between about 7.0 to about8.2, between about 7.0 to about 8.0, between about 7.5 to about 9.0,between about 7.5 to about 8.5, between about 7.5 to about 8.2, betweenabout 7.5 to about 8.2, between about 7.8 to about 9.0, between about7.8 to about 8.5, or between about 7.8 to about 8.2, etc. The buffer mayfurther contain one or more salt(s), in addition to any buffering salts.Ideally, a salt type and concentration will be used such thatRNA-protein interactions are weakened while maintaining the desired RNAin solution. For example, a total salt concentration of between 150 mMand 500 mM, or between 200 and 300 mM may be used. Preferably, the saltconcentration is 250 mM. The salt may be sodium chloride.

For example, the buffer may contain one or more salt at a total saltconcentration of between 0-500 mM, such as about 50 mM, about 75 mM,about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM,about 350 mM, about 400 mM, about 450 mM, about 500 mM, from about 50 mMto about 500 mM, from about 50 mM to about 400 mM, from about 50 mM toabout 300 mM, from about 50 mM to about 250 mM, from about 75 mM toabout 500 mM, from about 75 mM to about 400 mM, from about 75 mM toabout 300 mM, from about 75 mM to about 250 mM, from about 100 mM toabout 500 mM, from about 100 mM to about 400 mM, from about 100 mM toabout 300 mM, or from about 100 mM to about 250 mM, etc.

However, the inventors have found that excessive salt concentration inthe buffer should ideally be avoided due to the risk of RNAprecipitation during TFF or disadvantageous effects in any downstreammethods. It is therefore preferred that no salt, other than bufferingsalts, is added to the buffer for TFF purification. Addition of EDTA toa buffer is known to advantageously inhibit any RNase activity. However,methods of the invention can purify RNA without the addition of EDTA, sothe buffer may therefore be free from EDTA.

The volume ratio of the additional fluid (i.e. the fluid which is addedbeyond that of the sample) may influence the efficiency of the removalof small molecules during RNA purification and/or buffer exchange.However, larger volumes increase the operation time. Typically, thevolume ratio of the additional fluid to that of the sample is between5:1 and 10:1. The inventors have found that a ratio of about 8:1 isparticularly advantageous to ensure efficient purification and/or bufferexchange without unduly increasing the operation time.

Hydroxyapatite Chromatography

The inventors have devised an industrially scalable process that isparticularly useful for the purification of large RNA from IVT mixturesusing hydroxyapatite chromatography, but can also be used for thepurification of short (e.g. siRNA) and medium RNA (e.g. mRNA). Thus amethod of the invention can comprise one or more steps of hydroxyapatitechromatography.

A particular advantage of this technique is that a step comprisingenzymatic digestion of the template DNA can be omitted. This constitutesan improvement over prior art methods which rely on digestion of thetemplate DNA. This method is particularly advantageous for efficientlyremoving template-derived DNA or fragments thereof, as well as proteins,from the desired RNA species.

Hydroxyapatite chromatography involves hydroxyapatite as stationaryphase. Hydroxyapatite is a form of calcium phosphate having the chemicalformula [Ca₅(PO₄)₃(OH)]₂. Hydroxyapatite chromatography of nucleic acidsis believed to exploit the charge interaction between their negativelycharged phosphate backbone and the positively charged calcium ions onthe surface of the hydroxyapatite medium. Differential elution (e.g. toseparate protein, DNA and undesired RNA species from desired RNAspecies) is accomplished by the application of an increasing phosphategradient. Phosphate ions present in the buffer compete with thephosphate groups of the retained nucleic acid species for calcium on thehydroxyapatite medium, thus allowing separation by selective elution ofmolecules. In this mixed mode chromatography, the binding is a balanceof attraction of the RNA phosphate backbone to the calcium ions of thehydroxyapatite medium and repulsion of the RNA phosphate backbone fromthe phosphate of the hydroxyapatite medium. Compared to ion exchangechromatography, the strength of the binding on a hydroxyapatite mediumis dependent on charge density rather than total charge. This importantdifference allows for the separation of molecules upon their chargedensity (e.g. RNA vs DNA vs proteins) and the binding and elution of RNAregardless of its total charge, and therefore regardless of its length.Therefore this method can be used for the purification of RNA moleculesof any length.

The fractionation of nucleic acids using hydroxyapatite was described inthe 1960s (Bernardi et al. 1965). This approach has been exploited forapplications including isolation and separation of viral RNA, dsDNA andssDNA from environmental samples (Andrews-Pfannkoch et al. 2010),separation of DNA and RNA from tissue-extracted nucleic acids (Beland etal. 1979) and separation of DNA for hybridization studies (Kamalay etal. 1984). To the best knowledge of the inventors, there is no publishedevidence of the use of hydroxyapatite chromatography in a bioprocess forthe purification of RNA obtained from IVT, which poses specificchallenges to the skilled person due to the characteristics of thesample.

Hydroxyapatite chromatography parameters will be selected such that adesired RNA can be retained, and then selectively eluted, withoutsignificantly affecting RNA integrity and/or potency.

Hydroxyapatite chromatography may be performed using a batch format or acolumn format. A column format is preferred. The column comprises thestationary phase. Purification using a column format may includeapplying an RNA-containing sample to the column, discarding theflow-through, passing elution buffer through the column, and collectingthe desired eluates or fractions thereof. The method may compriseadditional steps such as wash steps before or during these steps.Suitable chromatography setups are known in the art, for example liquidchromatography systems such as the ÄKTA liquid chromatography systemsfrom GE Healthcare.

A preferred hydroxyapatite stationary phase is ceramic hydroxyapatite.Ceramic hydroxyapatite is a spherical, porous form of crystallinehydroxyapatite and is typically obtained by sintering crystallinehydroxyapatite at high temperatures. Hydroxyapatite chromatography usingceramic hydroxyapatite as stationary phase is particularly advantageousfor RNA purification a large-scale, as it is a particularly stablematerial that can withstand high flow rates and repeated use.

The nominal pore diameter of the hydroxyapatite particles is typicallybetween 0.05-0.13 μm, for example 0.08-0.1 μm.

The nominal mean particle size is typically 20-80 μm, for example 40 μm.

An exemplary hydroxyapatite medium is CHT™ Ceramic Hydroxyapatite fromBio-Rad (Type II, 40 μm particle size).

The chromatography is typically performed at a linear flow rate of250-350 cm/h, e.g. at 300 cm/h. Eluate fractions containing RNA may beidentified by measuring UV absorption at 260 nm. The compositioncomprising the RNA of interest collected in the eluate is highlypurified relative to the preparation before the hydroxyapatitechromatography step. Multiple eluted fractions containing the RNA ofinterest may be combined before further treatment.

Any suitable phosphate buffer may be used for elution. A particularlypreferred phosphate buffer is one which minimises the levels of RNAprecipitation compared to less preferred phosphate buffers when used atthe same concentration and pH. The inventors have found that a potassiumphosphate buffer is particularly suitable, and is preferred over asodium phosphate buffer because it advantageously minimises the levelsof RNA precipitation compared to a sodium phosphate buffer when used atthe same concentration and pH. Generally, the inventors have found thatusing cations with increasing kosmotropicity are preferred.

The inventors have also found that hydroxyapatite chromatographyparameters can be varied such that an improved separation of RNA and DNAis possible. This may be achieved by using an amount of a salt, inaddition to the phosphate salt(s), in one or more of the elutionbuffer(s) such that the concentration of the salt in the final elutionbuffer remains constant throughout elution. Any suitable salt may beused, for example sodium chloride. Additionally or alternatively, anamount of a salt may be added to the sample before the sample is appliedto the hydroxyapatite column. For example, potassium chloride or sodiumchloride may be added to the sample to a final concentration of between100-500 mM, e.g. 250 mM or 500 mM, and no salt is added to the phosphateelution buffer(s), provides a particularly advantageous method forpurification of RNA with high yield while maintaining a high degree ofpurity.

For example, sodium chloride and/or potassium chloride may be added tothe sample at a final concentration of between 0-500 mM, such as about50 mM, about 75 mM, about 100 mM, about 150 mM, about 200 mM, about 250mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500mM, from about 50 mM to about 500 mM, from about 50 mM to about 400 mM,from about 50 mM to about 300 mM, from about 50 mM to about 250 mM, fromabout 75 mM to about 500 mM, from about 75 mM to about 400 mM, fromabout 75 mM to about 300 mM, from about 75 mM to about 250 mM, fromabout 100 mM to about 500 mM, from about 100 mM to about 400 mM, fromabout 100 mM to about 300 mM, from about 100 mM to about 250 mM, etc.

The inventors have also found that, surprisingly, selective elution ofRNA but not DNA or other impurities may be achieved by using an elutionstep wherein the phosphate concentration in the elution buffer is suchthat RNA is selectively eluted. The exact range of a suitable phosphateconcentration may be determined empirically and depends on the presenceof any additional non-phosphate salts in the elution buffer, such assodium chloride. For example, where no additional salt is present, theinventors have found that using an elution step wherein the phosphateelution buffer has a conductivity of between about 1.8 S/m (18 mS/cm)and 3.8 S/m (38 mS/cm), or between 2.1 S/m (21 mS/cm) and 3 S/m (30mS/cm), for example about 2.1 S/m (21 mS/cm) results in selectiveelution of RNA.

For example, the phosphate elution buffer may have a conductivity ofabout 1.8 S/m (18 mS/cm), about 1.9 S/m (19 mS/cm), about 2.0 S/m (20mS/cm), about 2.1 S/m (21 mS/cm), about 2.2 S/m (22 mS/cm), about 2.3S/m (23 mS/cm), about 2.4 S/m (24 mS/cm), about 2.5 S/m (25 mS/cm),about 2.6 S/m (26 mS/cm), about 2.7 S/m (27 mS/cm), about 2.8 S/m (28mS/cm), about 2.9 S/m (29 mS/cm), about 3.0 S/m (30 mS/cm), about 3.1S/m (31 mS/cm), about 3.2 S/m (32 mS/cm), about 3.3 S/m (33 mS/cm),about 3.4 S/m (34 mS/cm), about 3.5 S/m (35 mS/cm), about 3.6 S/m (36mS/cm), about 3.7 S/m (37 mS/cm), about 3.8 S/m (38 mS/cm), from about1.8 S/m (18 mS/cm) to about 3.8 S/m (38 mS/cm), from about 1.8 S/m (18mS/cm) to about 3.5 S/m (35 mS/cm), from about 1.8 S/m (18 mS/cm) toabout 3.0 S/m (30 mS/cm), from about 1.8 S/m (18 mS/cm) to about 2.5 S/m(25 mS/cm), from about 1.8 S/m (18 mS/cm) to about 2.1 S/m (21 mS/cm),from about 2.0 S/m (20 mS/cm) to about 3.8 S/m (38 mS/cm), from about2.0 S/m (20 mS/cm) to about 3.5 S/m (35 mS/cm), from about 2.0 S/m (20mS/cm) to about 3.0 S/m (30 mS/cm), or from about 2.0 S/m (20 mS/cm) toabout 2.1 S/m (21 mS/cm), etc.

This can be achieved for example by using an elution step using anelution buffer having a concentration of about 180 mM potassiumphosphate. Suitable concentrations of other phosphate buffers (e.g.sodium phosphate) can also be used. Using a step-wise (non-continuous)elution gradient, comprising a step of selective RNA elution asdescribed, is particularly advantageous for the purification of RNA.

The addition of a salt to the sample and/or elution buffer(s) and theuse of a step-wise elution gradient, as mentioned above, can usefully becombined to obtain a particularly efficient separation of RNA fromimpurities. For example, the method may include using an amount of asalt, in addition to the phosphate salt(s), in one or more of theelution buffer(s) such that the concentration of the salt in the finalelution buffer remains constant throughout elution, or adding an amountof a salt to the sample before applying the sample to the hydroxyapatitecolumn (and optionally wherein no salt is added to the phosphate elutionbuffer(s)), and wherein the elution comprises an elution step whereinthe phosphate concentration in the elution buffer is such that RNA isselectively eluted.

Preferably, hydroxyapatite chromatography is used according to theinvention in combination with other RNA purification methods, forexample hydroxyapatite chromatography may be preceded by a method thatefficiently removes free nucleotides, because the inventors havesurprisingly found that these can block or saturate the column. Theinventors have found that such a combination of methods results inparticularly efficient purification of large RNA from an in vitrotranscription sample. For example, core bead flow-through chromatographyor tangential flow chromatography may be used as a purification steppreceding hydroxyapatite chromatography.

Core Bead Flow-Through Chromatography

According to the invention, RNA may be purified using core beadflow-through chromatography. Thus a method of the invention can compriseone or more steps of core bead flow-through chromatography. Theinventors have found that this technique enables a fast,industrial-scale purification process for obtaining pure RNA with highyield, and is particularly advantageous for removing proteincontaminants from a desired RNA species e.g. in an IVT reaction sample.The inventors have shown that very large RNA species comprising morethan 3 megadaltons may be purified using this method. To the bestknowledge of the inventors, there are no prior art methods that enablethe purification of such large RNA species, in particular after IVT,using core bead chromatography or any other methods. However, thismethod is not limited to the purification of large RNA molecules, andRNA molecules of any size (e.g. medium RNAs) can be purified with thismethod as long as a suitable bead pore size is selected, as describedbelow.

Core bead flow-through chromatography may be performed using a batchformat or a column format. A column format is preferred. The columncomprises the stationary phase. The column format may include applying aRNA-containing sample to the column, collecting the flow-through, andoptionally passing elution buffer through the column, and collecting thedesired eluates or fractions thereof. The method may comprise additionalsteps such as wash steps e.g. after applying the sample to the column, a“chase” buffer is usually added to the column. Suitable chromatographysetups are known in the art, for example liquid chromatography systemssuch as the ÄKTA liquid chromatography systems from GE Healthcare.

After applying the RNA-containing sample to the column, its contents cantravel through the column by gravitational force alone or externalpressure may be applied to increase the rate of their passage. Followingapplication of the RNA-containing sample to the column, a buffer mayalso be applied to the column, typically called a “chase buffer” in theart, and passed through the column using gravitational force alone or byapplying external pressure in order to increase the rate at which thesample components pass through the column. The flow rate can be statedas volumetric flow rate (volume of mobile phase, e.g. sample and/orchase buffer, passing through the column per unit time) or linear flowrate (distance of mobile phase front travelled per unit time). Methodsto calculate the flow rate and convert from linear to volumetric flowrate are known in the art.

According to the invention, the chromatography medium is comprised ofbeads that are comprised of a porous material (matrix), usually formedfrom a polymer. The matrix comprises at least two layers, for example aninner layer (core) surrounded by an outer layer (shell), but the matrixmay also comprise one or more additional (intermediate) layers betweenthe inner layer and the outer layer.

Each matrix layer may be functionalised with at least one ligand, or itmay not be functionalised. Typically, the layers can be distinguishedfrom each other by the presence or absence of at least one ligand.

For example, the core may be functionalised with N different ligands,whereas the shell is functionalised with no more than N−1 of theseligands. N may be any positive integer, for example 1. For example, thecore may be functionalised with a ligand whereas the shell isfunctionalised with one or more different ligands, or may not befunctionalised with any ligand. In a preferred embodiment, the core isfunctionalised with a ligand, whereas the shell is not functionalisedwith any ligands.

Preferably, at least one ligand is a ligand that has multiplefunctionalities, for example the ligand is both hydrophobic andpositively charged. For example, the ligand may be amono-(C1-C8)alkyl-amine, for example the ligand may be octylamine(CH₃(CH₂)₇NH₂).

Thus in a preferred embodiment of the invention, the core isfunctionalised with a ligand, wherein the ligand has multiplefunctionalities, for example the ligand is both hydrophobic andpositively charged, for example the ligand may be amono-(C1-C8)alkyl-amine, for example the ligand may be octylamine, andthe shell is not functionalised with any ligands.

The matrix has a defined pore size and thereby prevents a proportion ofmolecules from entering the core based on the size of the molecules,which are collected in the column flow-through (flow-through mode).Molecules that are able to pass through the matrix enter the core, wherethey may be retained, typically by binding to a ligand. Retainedmolecules may be eluted from the beads using a suitable eluent(bind-elute mode). Typically, the eluent is a solution comprising sodiumhydroxide (NaOH) and a solvent.

Core bead flow-through chromatography parameters will be selected suchthat a desired RNA can be selectively recovered from one or more of theflow-through fraction(s), without significantly affecting RNA integrityand/or potency.

Preferably, the matrix is a porous matrix, for example agarose,preferably a highly cross-linked agarose.

The matrix pore size is usually stated in kDa and refers to the averagemolecular mass of the smallest particle the matrix is likely to reject(also referred to as MWCO). Alternatively, the matrix pore size can bestated in μm and refers to the diameter of the smallest particle thematrix is likely to reject, as described above for TFF. The pore size isselected so that the cut-off is below RNA size but above protein size.Using this method, RNA species may be purified that ARE larger than themolecular cut-off of the beads. More preferably, the desired RNA speciesis the largest molecule in the sample to be purified. Therefore,according to this invention RNA is recovered from one or more of theflow-through fraction(s).

The inventors have found that for the purification of large RNAs aMWCO/pore size of at least 250 kDa is useful e.g. at least 300 kDa, 400kDa, 500 kDa, 600 kDa, or at least 700 kDa etc. A molecular weightcut-off of at least about 700 kDa is particularly preferred. Generally,the MWCO is selected such that the ratio of the molecular weight of theRNA molecule of interest to the MWCO is at least 1.5:1 (e.g. at least2:1, at least 3:1, at least 4:1, at least 5:1 or at least 6:1) and/orthat the ratio of the molecular weight of the largest non-RNA impurityto the MWCO is at least 1:1.5 (e.g. at least 1:2, at least 1:3, at least1:4, at least 1:5 or at least 1:6).

The average diameter (particle size) of the beads will be selected so toenable efficient RNA purification with minimal operation time withoutsignificantly affecting RNA integrity and/or potency due to excessivepressures required for performance. Larger particles and larger porestypically allow the use of lower pressures, but the separationefficiency may be reduced. The inventors have found that a particle sizeof about 50-100 μm is preferable, wherein a particle size of about 60-90μm is more preferably, and wherein a particle size of about 70-80 μm iseven more preferable. A particle size of about 85 μm is most preferred.

An exemplary core bead flow-through chromatography medium is Capto™ Core700 beads from GE Healthcare.

RNA is selectively recovered from the column in the flow-through.Proteins and short nucleic acids are retained in the beads. Flow-throughfractions containing RNA may be identified by measuring UV absorption at260 nm. The composition comprising the RNA of interest collected in theflow-through is highly purified relative to the preparation before thecore bead chromatography step. Multiple eluted fractions containing theRNA of interest may be combined before further treatment.

An amount of a salt may be added to the RNA-containing sample before thesample is passed through the column. The inventors have found that thisis particularly advantageous for the removal of protein impurities. Anysuitable salt may at a suitable concentration be used, for example atbetween about 150 mM and 500 mM.

For example, a salt may be added to the RNA-containing sample at a finalconcentration of between 0-500 mM, such as about 50 mM, about 75 mM,about 100 mM, about 125 mM, about 150 mM, about 200 mM, about 250 mM,about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM,about 600 mM, about 700 mM, about 750 mM, from about 50 mM to about 600mM, from about 50 mM to about 550 mM, from about 50 mM to about 500 mM,from about 50 mM to about 400 mM, from about 100 mM to about 600 mM,from about 100 mM to about 550 mM, from about 100 mM to about 500 mM,from about 100 mM to about 400 mM, from about 150 mM to about 600 mM,from about 150 mM to about 550 mM, from about 150 mM to about 500 mM,from about 150 mM to about 400 mM, etc.

The inventors have found that a salt concentration of between about 125mM and 250 mM is particularly advantageous, resulting in RNApurification with a high RNA yield and efficient protein removal.Alternatively, where a high RNA yield is required more than removal ofprotein impurities, for example where a sample that is substantiallyfree from protein is used, a salt concentration of not more than 250 mM,or preferably not more than 125 mM can be used. Where a high RNA yieldand/or high efficiency of nucleotide removal is required more thanremoval of protein impurities, for example where a sample that issubstantially free from protein but contains large amounts of freenucleotides is used, a salt concentration of not more than 250 mM,preferably not more than 125 mM, and most preferably no salt is added.

A suitable salt is typically a salt which minimises the levels of RNAprecipitation compared to less preferred salts when used at the sameconcentration and pH. The inventors have found that typically potassiumphosphate and/or potassium chloride are particularly suitable, andpreferred over sodium phosphate and sodium chloride, because potassiumsalts advantageously minimises the levels of RNA precipitation comparedto sodium salts when used at the same concentration and pH. Generally,the inventors have found that using cations with increasingkosmotropicity is preferred.

The RNA-containing sample may be diluted before the sample is passedthrough the column. For example, the sample may be diluted with adiluent volume that corresponds to about 5-fold, about 4-fold, about3-fold, about 2-fold, or about 1-fold of sample volume. A 1-folddilution means that a volume of a diluent that is equal to the volume ofthe sample is added to the sample. Any suitable diluent may be used andwill typically be a buffer. A suitable diluent is one that does notinterfere with any subsequent purification or buffer exchange steps. Forexample, the diluent may be a buffer that is the same as the buffer ofthe RNA-containing sample (e.g. 50 mM Tris, pH 8.0).

The flow rate may be varied to achieve improved RNA recovery and/orprotein removal. A linear flow rate of between 200 and 500 cm/h isadvantageous where a high RNA recovery is desired. A flow rate ofbetween 50 and 300 cm/h is advantageous where a high level of proteinremoval is desired. Typically, a flow rate of between 250 and 300 cm/h,preferably of about 275 cm/h is used for optimised recovery and proteinremoval.

The addition of a salt, dilution of the sample, and variation of theflow rate, as described above, can usefully be combined. For example,the RNA-containing sample may be diluted and an amount of a salt may beadded to the sample before the sample is passed through the column. Aparticularly advantageous method for the purification of large RNA withhigh purity, yield and short operation times is one where sample isdiluted 4-fold before applying the sample to the column, thechromatography is performed at a linear flow rate of 275 cm/h and saltis added to the sample and/or chase buffer at 250 mM (e.g. KCl or NaCl).

Where core bead chromatography is used according to the invention, it isparticularly useful for removing protein contaminants from an RNA ofinterest. Particularly good results are achieved where theRNA-containing sample that is applied to the chromatography column in asingle purification run contains no more than 5-15 mg total protein perml of stationary phase (i.e. core beads), e.g. no more than 10 mg/ml orno more than 13 mg/ml. These values are particularly relevant where thetotal protein is composed of proteins that are typically components ofan in vitro transcription reaction, such as T7 polymerase, cappingenzyme, RNase inhibitor and pyrophosphatase.

Where large-scale purification is performed, chromatography columns maybe connected to each other in series for increased capacity.

The inventors have also found that even higher levels of purity (e.g. bymore efficiently removing free nucleotides remaining from IVT) can beachieved where a step of core bead flow-through chromatography isfollowed by a step of TFF. The TFF step can be used to simultaneouslyperform RNA purification, in particular nucleotide removal, and bufferexchange by using a formulation buffer during the purification/bufferexchange process. The formulation buffer is different to thepurification buffer used in any preceding steps.

Combination of Methods

Any of the disclosed methods can be used in isolation or combined in aprocess comprising at least one step of RNA purification, and optionallya further step of buffer exchange. For example, TFF, core beadflow-through chromatography, or hydroxyapatite chromatography is usedfor RNA purification, optionally followed by a further step of TFF forbuffer exchange and/or RNA purification.

In another example, TFF is used in combination with hydroxyapatitechromatography for RNA purification, optionally followed by a furtherstep of TFF for buffer exchange and/or RNA purification.

In another example, core bead flow-through chromatography is used incombination with hydroxyapatite chromatography for RNA purification,optionally followed by a step of TFF for buffer exchange and/or RNApurification.

In another example, TFF is used in combination with core beadflow-through chromatography for RNA purification, optionally followed bya further step of TFF for buffer exchange and/or RNA purification.

In another example, where core bead flow-through chromatography is used,it is followed by tangential flow filtration. Such a method may alsoinclude hydroxyapatite chromatography which follows core beadflow-through chromatography and precedes tangential flow filtration.

In another example, where hydroxyapatite chromatography is used, it ispreceded by tangential flow filtration or core bead flow-throughchromatography. Such a method may also include tangential flowfiltration which follows hydroxyapatite chromatography.

Where a combination of methods comprises two steps of the same method,e.g. one step of tangential flow filtration and a further step oftangential flow filtration, two different buffers are typically used inthe first step and in the second step. Typically, the first buffer isdifferent from the second buffer in at least one component and/orcharacteristic. For example, the salt concentration, type of salt,tonicity, pH or amount of contaminants may be different. Usually, thebuffers will be based on different buffering systems, for example thefirst buffer may be a Tris or phosphate-based buffer, whereas the secondbuffer is a citrate-based buffer.

The inventors have shown that a combination of methods as recited aboveleads to even greater advantages in terms of purity (e.g. removal ofribonucleoside triphosphates, proteins, DNA and fragments thereof) andyield of the final RNA product, ease of operation, time efficiency andscalability of the overall process.

Apparatus Characteristics

One advantage of the invention is that it uses components which aredisposable. Thus a method of the invention can include a step in whichsome or all of the apparatus in which the method is performed arediscarded after the method is performed. For instance, any TFF columns,hydroxyapatite supports, and/or core bead flow-through columns can bediscarded, as can any tubing and connectors which were used to connectthem. These components may be discarded as biohazardous waste.

Thus methods of the invention can use disposable apparatus components.Furthermore, methods of the invention will, in general, use apparatuscomponents that can readily be decontaminated from RNase. Thisrequirement can be reflected in the materials, shape, configuration anddimensions of the components.

Quick Methods

As mentioned above, the invention provides a method for purifying RNAfrom a sample, wherein the RNA is purified to at least 99% purity inless than 12 hours.

Similarly, the invention provides a method for purifying RNA from asample which contains RNA, DNA, pyrophosphates, and free nucleotides (asun-reacted RNA precursors and/or as degradation products from RNA orDNA), wherein the method provides final material in less than 12 hourswhich is free from DNA, pyrophosphates, and free nucleotides.

The RNA-containing sample can be the product of an IVT reaction, and sowill contain the typical IVT contaminants discussed above.

The RNA can be prepared to a high purity e.g. ≧99.5%, ≧99.9%, or even≧99.95%. Thus at least 99% or more of the components in the purifiedmaterial (other than water and buffer salts) are a RNA of interest.

The method can be completed, to provide the purified RNA, in less than12 hours e.g. <8 hours, <6 hours, <4 hours, or <2 hours.

The method can use any of the steps (and combinations thereof) disclosedelsewhere herein. The method is ideally performed using aqueousconditions throughout.

Pharmaceutical Compositions

RNA purified according to this invention is useful as a component inpharmaceutical compositions, for example for use as a vaccine inimmunising subjects against various diseases. These compositions willtypically include RNA and a pharmaceutically acceptable carrier. Athorough discussion of pharmaceutically acceptable carriers is availablein Gennaro et al. A pharmaceutical composition of the invention can alsoinclude one or more additional components such as small moleculeimmunopotentiators (e.g. TLR agonists). A pharmaceutical composition ofthe invention can also include a delivery system for the RNA e.g. aliposome, an oil-in-water emulsion, or a microparticle.

A pharmaceutical composition of the invention is preferablysubstantially free from contaminants resulting from RNA manufacture andpurification. Where IVT is used, such contaminants may include proteins,e.g. enzymes such as polymerase, in particular T7 polymerase, andcapping enzymes, free nucleotides, and template DNA &/or fragmentsthereof.

Pharmaceutical compositions of the invention may include the RNA inplain water (e.g. w.f.i.) or in a final formulation buffer e.g. aphosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, ahistidine buffer, or a citrate buffer. Buffer salts will typically beincluded in the 5-20 mM range.

Compositions of the invention may include metal ion chelators. These canprolong RNA stability by removing ions which can acceleratephosphodiester hydrolysis. Thus a composition may include one or more ofEDTA, EGTA, BAPTA, pentetic acid, etc. Such chelators are typicallypresent at between 10-500 μM e.g. 0.1 mM. A citrate salt, such as sodiumcitrate, can also act as a chelator, while advantageously also providingbuffering activity.

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical e.g. about 9 mg/ml.

Pharmaceutical compositions of the invention may have a pH between 5.0and 9.5 e.g. between 6.0 and 8.0.

Pharmaceutical compositions of the invention may have an osmolality ofbetween 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, orbetween 290-310 mOsm/kg.

Pharmaceutical compositions of the invention may include one or morepreservatives, such as thiomersal or 2-phenoxyethanol. Mercury-freecompositions are preferred, and preservative-free vaccines can beprepared. Pharmaceutical compositions of the invention are preferablysterile.

Pharmaceutical compositions of the invention are preferablynon-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure)per dose, and preferably <0.1 EU per dose.

Pharmaceutical compositions of the invention are preferably gluten free.

Pharmaceutical compositions of the invention may be prepared in unitdose form. In some embodiments a unit dose may have a volume of between0.1-1.0 ml e.g. about 0.5 ml.

The compositions may be prepared as injectables, either as solutions orsuspensions. The composition may be prepared for pulmonaryadministration e.g. by an inhaler, using a fine spray. The compositionmay be prepared for nasal, aural or ocular administration e.g. as sprayor drops. Injectables for intramuscular administration are typical.

Where a composition includes a delivery system, this will usually be aliposome (e.g. see WO2012/006376, WO2012/030901, WO2012/031043,WO2012/031046, and WO2013/006825), an oil-in-water emulsion (e.g. seeWO2012/006380, WO2013/006834, and WO2013/006837), or a microparticle(e.g. see WO2012/006359). A process of the invention may include afurther step of combining a purified RNA molecule with a deliverysystem. Similarly, the invention provides a method for preparing apharmaceutical composition, comprising steps of: purifying a RNA using amethod of the invention; and combining the purified RNA with a deliverysystem e.g. with a liposome or with an oil-in-water emulsion.

Compositions comprise an immunologically effective amount of RNA, aswell as any other components, as needed. By ‘immunologically effectiveamount’, it is meant that the administration of that amount to anindividual, either in a single dose or as part of a series, is effectivefor treatment or prevention. This amount varies depending upon thehealth and physical condition of the individual to be treated, age, thetaxonomic group of individual to be treated (e.g. non-human primate,primate, etc.), the capacity of the individual's immune system tosynthesise antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials. The RNA content of compositions of the invention willgenerally be expressed in terms of the amount of RNA per dose. Apreferred dose has ≦100 μg RNA (e.g. from 10-100 μg, such as about 10μg, 25 μg, 75 μg or 100 μg), but expression can be seen at much lowerlevels e.g. ≦1 μg/dose, ≦100 ng/dose, ≦10 ng/dose, ≦1 ng/dose, etc.

The invention also provides a delivery device (e.g. syringe, nebuliser,sprayer, inhaler, dermal patch, etc.) containing a pharmaceuticalcomposition of the invention. This device can be used to administer thecomposition to a vertebrate subject.

Methods of Treatment and Medical Uses

Pharmaceutical compositions of the invention can be used in vivo foreliciting an immune response against an immunogen of interest.

The invention thus provides a method for raising an immune response in avertebrate comprising the step of administering an effective amount of apharmaceutical composition of the invention. The immune response ispreferably protective and preferably involves antibodies and/orcell-mediated immunity. The method may raise a booster response.

The invention also provides a pharmaceutical composition of theinvention for use in a method for raising an immune response in avertebrate.

The invention also provides the use of a pharmaceutical composition ofthe invention in the manufacture of a medicament for raising an immuneresponse in a vertebrate.

By raising an immune response in the vertebrate by these uses andmethods, the vertebrate can be protected against various diseases and/orinfections e.g. against bacterial and/or viral diseases as discussedabove. The compositions are immunogenic, and are more preferably vaccinecompositions. Vaccines according to the invention may either beprophylactic (i.e. to prevent infection) or therapeutic (i.e. to treatinfection), but will typically be prophylactic.

The vertebrate is preferably a mammal, such as a human or a largeveterinary mammal (e.g. horses, cattle, deer, goats, pigs). Where thevaccine is for prophylactic use, the human is preferably a child (e.g. atoddler or infant) or a teenager; where the vaccine is for therapeuticuse, the human is preferably a teenager or an adult. A vaccine intendedfor children may also be administered to adults e.g. to assess safety,dosage, immunogenicity, etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Thus a human patient may be less than 1 year old,less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old,or at least 55 years old. Preferred patients for receiving the vaccinesare the elderly (e.g. ≧50 years old, ≧60 years old, and preferably ≧65years), the young (e.g. ≦5 years old), hospitalised patients, healthcareworkers, armed service and military personnel, pregnant women, thechronically ill, or immunodeficient patients. The vaccines are notsuitable solely for these groups, however, and may be used moregenerally in a population.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,intradermally, or to the interstitial space of a tissue). Alternativedelivery routes include rectal, oral (e.g. tablet, spray), buccal,sublingual, vaginal, topical, transdermal or transcutaneous, intranasal,ocular, aural, pulmonary or other mucosal administration. Intradermaland intramuscular administration are two preferred routes. Injection maybe via a needle (e.g. a hypodermic needle), but needle-free injectionmay alternatively be used. A typical intramuscular dose is 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity,preferably to elicit an enhanced systemic and/or mucosal immunity.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In oneembodiment, multiple doses may be administered approximately 6 weeks, 10weeks and 14 weeks after birth, e.g. at an age of 6 weeks, 10 weeks and14 weeks, as often used in the World Health Organisation's ExpandedProgram on Immunisation (“EPI”). In an alternative embodiment, twoprimary doses are administered about two months apart, e.g. about 7, 8or 9 weeks apart, followed by one or more booster doses about 6 monthsto 1 year after the second primary dose, e.g. about 6, 8, 10 or 12months after the second primary dose. In a further embodiment, threeprimary doses are administered about two months apart, e.g. about 7, 8or 9 weeks apart, followed by one or more booster doses about 6 monthsto 1 year after the third primary dose, e.g. about 6, 8, 10, or 12months after the third primary dose.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to charge, to cations, to anions, to zwitterions, etc., aretaken at pH 7.

The term “yield” in the context of this invention stands for thefraction of RNA contained in sample after purification compared tobefore purification. Typically, yield is expressed as % yield,calculated according to the formula: [(amount RNApost-purification/amount RNA pre-purification)×100]. RNA amounts in asample can be measured using methods known in the art, for example usingan RNA-specific fluorescent dye such as RiboGreen®.

The term “purification” or “purify” means that a desired RNA in a sampleis separated from undesired components. “RNA purification” thus refersto methods for purifying a RNA of interest from a composition comprisingthe RNA of interest and impurities. Thus, after purification the RNA ispresent in a purer form than before purification. This means thatundesired components are present at lower amounts relative to the amountof desired RNA than before purification. Undesired constituents ofRNA-containing samples which may need to be separated from the desiredRNA may include DNA, deoxynucleoside monophosphates, ribonucleosidetriphosphates, undesired RNA species (e.g. RNA that is longer/shorterthan a desired RNA size or outside a desired RNA size range, ordouble-stranded RNA vs single-stranded RNA), deoxy-oligonucleotides,proteins (in particular enzymes such as RNA polymerases e.g. T7polymerase, mRNA capping enzyme, pyrophosphatase, DNase, RNaseinhibitors), etc.

The words “potency” or “functionality” describe the intended biologicalfunction of the RNA molecule and the level to which that function isretained after purification compared to before purification of the RNA.For example, if the RNA potency remains unchanged after purification,then the extent of a particular biological function of the RNA has notchanged, for example as measured by the in vivo expression level of anyencoded protein relative to a certain amount of input RNA.

The word “stability” refers to the extent to which an RNA moleculeretains its structural integrity and resists degradation during physicalor chemical manipulations. For example, if RNA stability remainsunchanged after purification, then the level of structural integrity hasnot changed, for example measured by analysing the average RNA size orthe RNA size distribution.

The terms “preparative”, “large scale”, “commercial scale” and“industrial scale” in relation to a RNA purification method mean thatlarge quantities of RNA can be purified to a purity of at least 90%.Such large quantities are for example at least 0.5 mg, 1 mg, 2.5 mg, 5mg, 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, or even at least 1000mg using the method of the invention.

The term “stationary phase” refers to the non-mobile phase contained ina chromatographic bed.

The term “particle size” refers to the average diameter of a stationaryphase particle.

The term “pore size” refers to the average size of the smallest particlethat a stationary phase will reject or that a membrane will retain onthe sample side. The size is typically expressed in particle diameter ormolecular mass.

The term “elution gradient” means that the composition of the eluent isvaried throughout the elution process in a continuous or step-wisemanner. In contrast, “isocratic elution” proceeds using a fixed eluentcomposition throughout the elution process.

A “step” is different from another step of RNA purification or bufferexchange where the steps use different methods (e.g. tangential flowfiltration vs. hydroxyapatite chromatography) or where the steps use thesame method but are performed under different conditions (e.g. using adifferent buffer, a different membrane, or a different stationaryphase).

When a second step is performed “after” or “following” another firststep, the second step may be performed immediately after the previousfirst step in the method, i.e. no other step is performed between thefirst step and the second step, other than steps such as dilution orstorage which may take place in between the two steps. Alternatively,other step(s) may be performed between the first and second steps.

When a first step is performed “before” or “preceding” another secondstep, the first step may be performed immediately before the subsequentstep in the method, i.e. no other steps are performed between the firststep and the second step, other than steps such as such as dilution orstorage which may take place in between the two steps. Alternatively,other step(s) may be performed between the first and second steps.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the result of RNA purification using tangential flowfiltration and hydroxyapatite chromatography: in vitro transcriptionreaction sample—protein removal—in vitro transcription reaction samplebefore purification (lane 1), after tangential flow filtration (lane 2),after hydroxyapatite chromatography (lane 3), after tangential flowfiltration (lane 4).

FIGS. 2A-2B show the result of RNA purification using hydroxyapatitechromatography. FIG. 2A illustrates Hofmeister series of ions in orderof their ability to salt out proteins. FIG. 2B shows the dynamic lightscattering analysis of RNA aggregate particle size in various elutionbuffers—x-axis: salt concentration in mM; y-axis: particle radius in nm.

FIG. 3A shows the result of RNA purification using tangential flowfiltration and hydroxyapatite chromatography: in vitro transcriptionreaction sample—protein removal—in vitro transcription reaction samplebefore purification (lane 1), after tangential flow purification (lane2), after hydroxyapatite chromatography (lane 3). FIG. 3B shows theresult of RNA purification using tangential flow filtration andhydroxyapatite chromatography: in vitro transcription reactionsample—DNA removal—DNA per mg of RNA present in an in vitrotranscription reaction sample before purification (first data point fromleft), after tangential flow filtration (second data point), afterhydroxyapatite chromatography (third data point).

FIG. 4A-4B show the result of RNA purification using core beadflow-through chromatography: in vitro transcription reactionsample—protein removal—in vitro transcription sample before purification(lane 1), flow-through after core bead flow-through chromatography (lane2), eluate after column cleaning-in-place (lane 3).

FIG. 5 shows the result of RNA purification using core bead flow-throughchromatography: in vitro transcription reaction sample—effect of salt insample and chase buffer on protein removal.

FIG. 6A shows the result of quantification of RNA or RNA plusnucleotides. FIG. 6B and FIG. 6D show the result of tangential flowfiltration vs. core bead flow-through chromatography—in vitrotranscription reaction sample—nucleotide removal. FIG. 6C shows theresult of tangential flow filtration vs. core bead flow-throughchromatography vs. core bead flow-through chromatography andhydroxyapatite chromatography vs. tangential flow filtration andhydroxyapatite chromatography—in vitro transcription reactionsample—protein removal.

FIGS. 7A-7G show the result of RNA purification using combination ofmethods described herein: in vitro transcription reaction sample—effecton RNA recovery, protein removal, nucleotide removal and DNA removal.FIG. 7A shows recovery of RNA measured by direct quantification byRiboGreen® assay (sample dilution 10,000 fold). FIG. 7B shows the purityof RNA sample by quantitative ELISA (T7 polymerase, samples in italicsare below LOQ). The graph shows detectable T7, below LOQ for mostsamples by ELISA. FIG. 7C shows the purity of RNA sample by quantitativeELISA (capping enzyme, samples in italics are below LOQ, “0” equalsbelow LOD). The graph shows capping enzyme below LOD in sample P3, postCC250 and P2 and P4, post HTP. FIG. 7D shows the purity of RNA sample bySDS page—silver staining (4 ug RNA loaded per lane). FIG. 7E showsnucleotide removal, expressed as ratio of quantification byOD/quantification by RiboGreen® (Y-axis refers to OD/RiboGreen®). FIG.7F shows plasmid DNA carryover, using qPCR assay on plasmid. DNA beforepurification: 1.0 ng/dose. Following purification: 0.6/0.7 ng/dose.Lowest concentration was found after HTP chromatography. Same TFFcartridge was used in all 4 processes/steps: possible carryover. Nobackground (buffer) control was used in this experiment. FIG. 7G showsthe level of E. coli protein contamination, using host (E. coli) proteinoat polyclonal antibodies (HRP labelled) to E. coli. In this assayL.O.D. (limit of detection) is ing/band, 4 ug RNA was loaded in eachlane. The figure shows that host contaminant below 1 ng/protein.

FIG. 8 shows the design of an experimental statistically significantapproach for core bead flow-through chromatography—in vitrotranscription reaction sample—optimisation of parameters—saltconcentration: 0 mM (−1) to 500 mM (1); sample dilution: no dilution (1)to 4-fold dilution (−1); flow rate (linear velocity): 50 cm/h (−1) to500 cm/h (1).

MODES FOR CARRYING OUT THE INVENTION Example 1 Method for QuantifyingRNA Yield and Nucleotide Removal

RNA was quantified in samples using an RNA-specific fluorescent dye(RiboGreen®). RNA levels before and after purification were compared tocalculate % RNA yield. RiboGreen® does not detect free nucleotides.

Free nucleotides are found in the unpurified in vitro transcription(IVT) reaction and include un-reacted precursors for RNA (ribonucleosidetriphosphate) and degradation products from DNAse digestion(deoxynucleosides monophosphate). A method was developed to measurenucleotides in the presence of RNA. Pure RNA was measured withRiboGreen® (FIG. 6A, 1^(st) bar) and by optical density (OD) at 260 nm,using 40 as a standard approximated extinction coefficient for RNA(2^(nd) bar). A mix of nucleotides was added to the pure RNA sample in aten-fold excess to RNA by mass. The resulting samples were measuredagain with RiboGreen® (3^(rd) bar) and by OD (4^(th) bar).

The results show that the measurement by RiboGreen® is unaffected by thepresence of nucleotides in the sample, while the detected OD valuesreflect the total concentration of RNA and nucleotides in the sample.The presence of nucleotides, as an indicator for nucleotide removalafter an RNA purification step, was calculated as the ratio of the ODmeasurement and the RiboGreen® assay measurement. A ratio ofapproximately 1 indicates pure RNA, i.e. complete nucleotide complete.Ratios above 1 indicate the presence of nucleotides in the sample.

Example 2 RNA Purification and Buffer Exchange Using Tangential FlowFiltration

A 10-kb RNA replicon was produced through in vitro transcription andcapping with completely chemical-defined enzymes, template, substanceand buffers. A KrosFlo Research IIi Tangential Flow Filtration Systemwas used (Spectrum Laboratories) for both RNA purification and bufferexchange in one single closed system. Various parameters were tested foroptimal results as indicated below: membrane chemistry, membrane poresize, membrane area, transmembrane pressure, shear rate (retentatevelocity), buffer volume, buffer capacity, buffer pH, sample saltconcentration, and the presence of EDTA in the buffer.

Parameters considered for Theoretical impact Conditions Conditionoptimization on RNA quality screened selected TFF cartridge MembraneInteraction of membrane mPES. PS mPES from chemistry with RNA andprotein (Spectrum and Spectrum RNA recovery and Watersep) proteinremoval Membrane Retain large MW particle 500 kD, 750 kD 500 kD MWCOpore size and remove small MW MWCO molecules 0.05 and 0.1 μm RNArecovery and protein removal Membrane area Buffer exchange 20, 52, 115cm² 115 cm² efficiency Operation time TFF system variables TMPRNA/protein separation 1-5 Psi 2 Psi (transmembrane Gel layer formationpressure) Shear rate RNA integrity 1000-5000 S⁻¹ ~800 S⁻¹ (retentatevelocity) Gel layer formation Dialysis buffer Small molecule removal5x-10x sample 8x sample volume volume Buffer exchange efficiency volumeoperation time Purification buffer Buffer capacity Buffer exchange 2, 10mM Citrate 10 mM Tris efficiency and 10, 50 mM Tris (buffer change fromRNA synthesis and to formulation) Buffer pH Interaction of RNA with pH6.5, 7.0, 7.5, 8 0, pH 8.0 protein 8.5 and 9.0 Protein aggregation Saltconcentration Interaction of RNA with 150, 250 and 500 mM 250 mM NaClprotein NaCl EDTA Interaction of RNA with 0, 1, 10, and 20 mM 0 mM RNAbinding protein Stability RNA stability

Four consistency runs were performed using the optimised conditions anddemonstrated that the tangential flow filtration method purifies RNAwith high recovery (>95%), purity as measured by protein removal (>90%of T7 RNA polymerase removed, as quantified by ELISA; 5 ng T7 polymeraseper 75 μg RNA post purification; >95% vaccinia capping enzyme removed,as quantified by ELISA) and as measured by nucleotide removal (>99.9% offree nucleotides removed, as quantified using the assay of Example 1),potency (no change in potency after purification) and stability (RNA isstable after purification). The operation as a single closed systemprevents contamination with exogenous agents such as RNase. The methodis quick (approx. 70 mins total) and easy to operate.

As shown in FIGS. 6B and 6D and FIG. 7E the method is particularlyuseful for removing free nucleotides from the sample. As shown in FIG. 5(lane 11), FIG. 6C and FIGS. 7B, 7C and 7D, protein impurities are alsoefficiently removed from the sample.

Example 3 RNA Purification Using Hydroxyapatite Chromatography

To test whether hydroxyapatite chromatography could be useful for thepurification of large RNA, 80 μg of lithium chloride purified 10-kb RNA(replicon) from an in vitro transcription reaction were loaded on ahydroxyapatite column and eluted with a phosphate linear gradientcomposed of varying proportions of Buffer A (10 mM phosphate buffer, pH6.8) and Buffer B (500 mM phosphate buffer, pH 6.8). It was found thatmRNA can be efficiently bound and recovered from a hydroxyapatitecolumn. RNA yield/recovery were measured by loading identical amounts oflithium chloride purified mRNA from an in vitro transcription reactionon a hydroxyapatite column or fed into the chromatography systemby-passing the column. Area under the elution peaks was calculated andthe ratio used as an indicator of RNA yield after column pass-throughcompared to without column pass-through purification (1401.25 mAu/ml vs1934.76 mAu/ml). The RNA yield was calculated as 72%. Lithium chloridepurified 10-kb RNA (replicon) from an in vitro transcription reactionwas loaded on a hydroxyapatite column and eluted using phosphate buffer.Collected fractions 4, 5 and 6 were loaded on a denaturing RNA gel,confirming that the optical density read is associated with RNA.

To test whether RNA can be more efficiently separated from contaminantssuch as protein or non-digested DNA using hydroxyapatite chromatography,the elution dynamics of purified RNA were analysed in the presence ofvarious amounts of a salt (0-1000 mM sodium chloride) in the elutionbuffer. Sodium chloride was added to both elution buffers A and B so tohave a constant concentration throughout the phosphate gradient. Therightward shift of the RNA elution peak shows that an increasingconcentration of phosphate is required for RNA elution with increasingsalt concentrations. This allows for the setup of different conditionsto further separate RNA from proteins or other impurities. The additionof salt to the phosphate elution buffer can therefore be exploited tooptimise fractionation of RNA from impurities. It was found that mRNAyield is inversely related to the concentration of sodium chloride inthe elution buffer.

To test whether RNA can be more efficiently separated from (undigestedtemplate) DNA, 100 μg of pure DNA or pure RNA were subjected tohydroxyapatite chromatography using the same parameters. A continuousgradient of a potassium phosphate elution buffer was used. Effect ofelution conditions on separating DNA from RNA was determined. It wasfound that DNA is eluted at higher phosphate concentrations than RNA(rightward shift of the elution peak). The inventors therefore devised astep-wise elution method whereby the phosphate concentration in theelution buffer increases step-wise, rather than continuously. RNA cantherefore be selectively eluted by choosing an elution buffer phosphateconcentration at which RNA but not DNA or other contaminants are eluted.A test run was then performed where equal amounts of purified RNA andDNA were mixed to a total amount of 200 μg in solution and subjected tohydroxyapatite chromatography using a step-wise elution gradient ofBuffer A and B.

In a gradient elution, RNA elution occurred at a buffer conductivity ofaround 21.04 mS/cm. DNA elution occurred at around 30.52 mS/cm. Thisdemonstrates that in the presence of an RNA/DNA mixture, a concentrationof about 180 mM potassium phosphate (or any potassium phosphateconcentration resulting in a conductivity value above 21.04 mS/cm andbelow 30.52 mS/cm) elutes selectively RNA and not DNA. A test run wasthen performed where purified DNA was analysed under the same conditionsas described above. No elution was observed below about 180 mM (˜18% B)potassium phosphate. The results show that RNA and DNA can efficientlybe separated with a step-wise elution. DNA elution can be achieved with38% buffer B, about 380 mM potassium phosphate (or any potassiumphosphate concentration resulting in a conductivity value above 30.52mS/cm). Using tangential flow filtration and hydroxyapatitechromatography (in vitro transcription reaction sample), elutionconditions for separating DNA from RNA were optimised.

In comparing various phosphate buffers useful for elution of RNA duringhydroxyapatite chromatography, it was found that a potassium phosphatebuffer performs better than a sodium phosphate in keeping RNA insolution and is a better candidate for hydroxyapatite column elution.Dynamic light scattering experiments (FIG. 2) showed that an increasingconcentration of sodium phosphate in the elution buffer duringhydroxyapatite chromatography leads to an increasingly larger apparentparticle size of the eluted RNA, probably due to salt-induced RNAprecipitation (“salting out”). This effect is reduced when usingpotassium phosphate instead of sodium phosphate at the sameconcentration, with concentrations up to 500 mM. A potassium phosphatebuffer was tested for RNA elution from a hydroxyapatite column andperformed comparably to a sodium phosphate buffer in terms of RNA purityand recovery for this process. Potassium phosphate is thereforeidentified as the salt of choice for RNA purification by hydroxyapatitechromatography.

Next, a non-purified in vitro transcription reaction containing 100 μgof a 10-kb RNA replicon was analysed using hydroxyapatitechromatography. Collected fractions 1, 2 and 3 were loaded on adenaturing RNA gel. No RNA was visible on the gel. Fractions B9(corresponding to fraction directly preceding fraction 2) and C1(corresponding to fraction 3) were analysed by reversed phase HPLC. Theelution time was compared to nucleotide standards, confirming that theobserved elution peaks at OD 260 using a non-purified in vitrotranscription reaction sample were mainly composed of free nucleotidesfrom the in vitro transcription reaction.

Example 4 RNA Purification Using Tangential Flow Filtration andHydroxyapatite Chromatography

A combination of tangential flow filtration followed by hydroxyapatitechromatography was tested for improved efficiency of RNA purificationfrom an in vitro transcription reaction sample, and in particular forthe removal of nucleotides before the sample is used in hydroxyapatitechromatography. An unpurified in vitro transcription reaction containinga 10-kb RNA replicon product was used as the starting sample. FIGS. 3Aand 3B show that such a combination of method allows the efficientremoval of nucleotides in the tangential flow filtration step and of DNA(reduced to 5.93 ng DNA per mg purified RNA) and protein (reduced tobelow detection levels) in the hydroxyapatite chromatography step,enabling the recovery of pure RNA (>80%) after the hydroxyapatitechromatography step (a step-wise elution gradient as described inExample 3 was used for elution). This is particularly useful as templateDNA digestion can be omitted from the overall RNA purificationprocedure, leading to faster operation times.

FIG. 1 further confirms the efficiency of protein removal using ahydroxyapatite chromatography step, showing that the level of proteinimpurities is reduced to below the level of detection using silverstaining (4 μg of purified RNA were loaded per lane). An optionalfurther step of tangential flow filtration was used to exchange thephosphate buffer in which the purified RNA is eluted followinghydroxyapatite chromatography into a citrate buffer suitable fordownstream applications.

FIG. 6C (lane “TFF0HTP0” vs. lane “Input”) also confirms the usefulnessof an RNA purification method combining tangential flow filtrationfollowed by hydroxyapatite chromatography for removing proteinimpurities from an RNA-containing sample.

Example 5 RNA Purification Using Core Bead Flow-Through Chromatography

Core bead flow-through chromatography was tested for the purification ofRNA. An unpurified in vitro transcription reaction (in Tris 50 mM, pH8.0) containing a 10-kb RNA replicon product was used as the startingsample. A HiScreen Capto™ Core 700 column (product code: 17-5481-15) wasinitially used, on a GE ÄKTAa Explorer 100 FPLC system. The sample wasdiluted in a buffer of Tris 50 mM, pH 8.0, to a final RNA concentrationof 600 ng/μl (final volume: 8.5 ml, containing 5.1 mg RNA). The samplewas injected into the column and chased with Tris buffer (50 mM) untilelution of the sample was complete. The flow was set at 1 ml/min(corresponding to 125 cm/h). Column cleaning-in-place (CIP) andregeneration was as per the manufacturer's instructions. It was foundthat RNA can be recovered in the column flow-through (e.g., in vitrotranscription reaction sample, 5.1 mg RNA, RNA was eluted in flowthrough). FIGS. 4A and 4B show that RNA is recovered from the columnflow-through at a high level of yield (FIG. 4B, lane 2 vs. lane 1) andcontains lower levels of protein impurities compared to before core beadflow-through chromatography (FIG. 4A, lane 2 vs. lane 1).

To test the effect of the presence of salt on removal of proteinimpurities using core bead flow-through chromatography, increasingconcentrations of sodium chloride or sodium phosphate added to thesample upon purification and in the chase buffer were tested.Chromatographic conditions for these purifications were equivalent tothe ones specified above. Flow-through fractions containing an equalamount of purified RNA (5 μg) were analysed by polyacrylamide gelelectrophoresis and silver staining. FIG. 5 shows that an increasingsalt concentration positively correlates with the level of removal ofproteinaceous contaminants. Salt was added to the sample and to thechase buffer. Arrows indicate two protein contaminants (T7 polymeraseand large subunit of the capping enzyme). Control sample on the farright is after purification of an in vitro transcription reaction usingtangential flow filtration only. In conclusion, increasing saltconcentration facilitates the removal of protein carryover, leading to afinal protein mass that is below the level of detection using asilver-stained polyacrylamide gel and 5 μg of RNA.

Conditions for core bead flow-through chromatography were furtheroptimised, in particular the salt concentration (0-500 mM), flow rate(50-500 cm/h), and sample dilution (4-fold dilution to undiluted; beforeapplication to the column) were varied and evaluated for their effect onthe level of RNA yield (recovery), protein removal (T7 polymerase and/orcapping enzyme) and nucleotide removal after core bead flow-throughchromatography and the pre-column pressure and operation time of thechromatography method. FIG. 8 shows the design of an experimentalstatistically significant approach. The value ranges for the testedvariables and the output parameters that were evaluated are indicated.Model details: Response Surface Designs, Central Composite Designs,Inscribed. The starting sample was an unpurified in vitro transcriptionreaction sample containing the RNA of interest. Protein carryover wasevaluated by silver-stained SDS page of the flow through material, andquantified by densitometry of the protein bands. Nucleotide removal andRNA yield were quantified as described in Example 1.

Table 1 shows the output values RNA yield (recovery), protein removal(T7 polymerase and capping enzyme), OD260 nm values, operation time andpre-column pressure after a core bead flow-through chromatography rununder different conditions (samples A-T).

TABLE 1 Inscribed CCI Samples Flow Salt Conc Flow cm/h Salt mM Conc injVol Inj RNA ug ul on GEL avg OD ng/ul A −0.594 −0.594 −0.594 141.3 101.50.402 500 106.0 19.9 91.9 B 0.594 −0.594 −0.594 408.7 101.5 0.402 500306.5 19.9 103.9 C −0.594 0.594 −0.594 141.3 398.5 0.402 500 106.0 19.9131.0 D 0.594 0.594 −0.594 408.7 398.5 0.402 500 306.5 19.9 142.9 E−0.594 −0.594 0.594 141.3 101.5 0.848 500 106.0 9.4 227.7 F 0.594 −0.5940.594 408.7 101.5 0.848 500 306.5 9.4 254.8 G −0.594 0.594 0.594 141.3398.5 0.848 500 106.0 9.4 259.6 H 0.594 0.594 0.594 408.7 398.5 0.848500 306.5 9.4 291.1 I 0.000 0.000 0.000 275.0 250.0 0.625 500 206.3 12.8204.0 L 0.000 0.000 0.000 275.0 250.0 0.625 500 206.3 12.8 206.1 M 0.0000.000 0.000 275.0 250.0 0.625 500 206.3 12.8 206.4 N 0.000 0.000 0.000275.0 250.0 0.625 500 206.3 12.8 211.8 O −1.000 0.000 0.000 50.0 250.00.625 500 37.5 12.8 180.8 P 1.000 0.000 0.000 500.0 250.0 0.625 500375.0 12.8 210.7 Q 0.000 −1.000 0.000 275.0 0.0 0.625 500 206.3 12.8140.3 R 0.000 1.000 0.000 275.0 500.0 0.625 500 206.3 12.8 208.2 S 0.0000.000 −1.000 275.0 250.0 0.250 500 206.3 32.0 72.0 T 0.000 0.000 1.000275.0 250.0 1.000 500 206.3 8.0 315.9 Ribogreen AU Flow through Precolumn Samples total recovery % ng/ul total recovery % Protein TimePressure A 229.6 95.2 112.8 281.9 116.8 0.162 0.0176 0.140 B 259.6 107.6119.3 298.2 123.6 0.297 0.0061 0.260 C 327.5 135.7 105.1 262.8 108.90.083 0.0176 0.140 D 357.1 148.0 102.3 255.7 106.0 0.100 0.0061 0.260 E569.3 111.9 214.8 537.1 105.6 0.180 0.0083 0.160 F 636.9 125.2 237.4593.5 116.7 0.221 0.0029 0.260 G 648.9 127.6 183.2 458.0 90.0 0.0800.0083 0.160 H 727.8 143.1 202.4 506.1 99.5 0.122 0.0029 0.280 I 509.9136.0 198.5 496.2 132.3 0.160 0.0058 0.200 L 515.1 137.4 191.6 479.0127.7 0.169 0.0058 0.200 M 516.0 137.6 160.7 401.6 107.1 0.144 0.00580.200 N 529.5 141.2 174.3 435.7 116.2 0.104 0.0058 0.200 O 452.0 120.5157.7 394.1 105.1 0.062 0.0320 0.070 P 526.8 140.5 197.7 494.3 131.80.112 0.0032 0.320 Q 350.6 93.5 178.1 445.1 118.7 0.156 0.0058 0.210 R520.4 138.8 142.1 355.2 94.7 0.064 0.0058 0.200 S 180.0 120.0 76.4 190.9127.3 0.018 0.0145 0.200 T 789.8 131.6 269.6 674.1 112.3 0.096 0.00360.200

The output parameters T7 polymerase removal and capping enzyme removalin samples A-T were quantified by resolution of the core beadchromatography flow through fraction using polyacrylamide gelelectrophoresis and silver-stained followed by quantification usingdensitometry of the protein bands. An unpurified in vitro transcriptionsample was used as control. Results were further analysed according tochromatography conditions: effect of salt concentration and sampledilution on RNA recovery, T7 polymerase removal (quantification inrelative units) and capping enzyme removal (quantification in relativeunits); effect of flow rate and sample dilution on RNA recovery, T7polymerase removal and capping enzyme removal; effect of flow rate andsalt concentration on RNA recovery, T7 polymerase removal and cappingenzyme removal.

Using an unpurified in vitro transcription reaction as the startingsample, the maximum sample volume per column volume (CV) was determinedat which protein is sufficiently removed using core bead flow-throughchromatography. Effect of sample-to-column volume ratio on proteinremoval was determined. Samples were diluted up to a maximum sample/CVratio of 10:1 (CV: 1 ml; ID: 0.7 cm; height: 2.5 cm, L. vel: 250 cm/h;flow: 1.6 ml/min; contact time: 36 seconds) or 64:1 (CV: 0.137 ml; ID:0.5 cm; height: 0.7 cm, L. vel: 250 cm/h; flow: 0.82 ml/min; contacttime: 10 seconds) and potassium chloride was added to a finalconcentration of 250 mM. The flow-through from each run was analysed bypolyacrylamide gel electrophoresis and silver staining. It was foundthat protein break-through occurred when the sample-CV ratio exceededabout 10:1, under the conditions used. In conclusion, a sample/CV ratioof up to 10 efficiently purified RNA from protein impurities in theexperimental condition used (e.g. 10 ml IVT reaction can be diluted to40 ml and efficiently purified with a 1 ml column).

Further, various sample and/or chase buffers compositions for use incore bead flow-through chromatography were compared with regards to thedegree of observed RNA precipitation in these buffers, measured usingdynamic light scattering and an increasing apparent particle size as anindicator of RNA precipitation. Table 2 summarizes the results of corebead flow-through chromatography: dynamic light scattering analysis ofRNA aggregate particle size in the presence of various salts. The secondcolumn refers to salt concentration in mM. Numbers in columns 3-7 areparticle radius in nm. The Table shows that potassium phosphate buffer(pH 6.5) and potassium chloride buffer (pH 8.0) are good candidates foran optimised flow through purification.

TABLE 2 Tris 10 mM Tris 10 pH 8.0 + mM pH 8.0 + KPO4 KPO4 NaPO4 NaCl KClpH 6.5 pH 8.0 pH 6.5 NaCl 0 20.3 (mM) 83 23.2 22.3 21.1 20.2 21.9 16721.8 20.4 20.1 19.8 22.2 250 22 19.7 20 20.7 23.8 333 23.9 19.4 20.723.1 26.8 417 27.6 20.2 21.3 26.6 31.8 500 32.6 21.3 22.5 31.1 39.8

Example 6 RNA Purification Using Core Bead Flow-Through Chromatographyand Tangential Flow Filtration

Using an unpurified in vitro transcription reaction as the startingsample containing a 10-kb RNA replicon product, nucleotide and proteinremoval were compared using either tangential flow filtration or corebead flow-through chromatography (using potassium chlorideconcentrations of 0, 250 or 500 mM in the sample). FIGS. 6B and 6D showthat tangential flow filtration efficiently removes nucleotideimpurities. FIG. 6C shows that core bead flow-through chromatographyefficiently removes protein impurities in the presence of potassiumchloride. Therefore, where it is desired to remove nucleotide andprotein impurities, it is desired that core bead flow-throughchromatography is followed by tangential flow filtration.

Example 7 RNA Purification Using Core Bead Flow-Through Chromatographyand Hydroxyapatite Chromatography

The presence of additional salts such as potassium chloride in thesample and/or chase buffer may sometimes be undesired. Using anunpurified in vitro transcription reaction as the starting samplecontaining a 10-kb RNA replicon, protein removal was compared using corebead flow-through chromatography (without additional salt, i.e. 0 mMpotassium chloride) alone or followed by hydroxyapatite chromatography(also without additional salt, i.e. 0 mM sodium chloride). FIG. 6C (lane“CC0HTP0” vs. lane “CC0”) shows that efficient protein removal can beachieved even in the absence of additional salt, when core beadflow-through chromatography is followed by hydroxyapatitechromatography.

Example 8 Combinations of Methods for RNA Purification and BufferExchange

Four different process streams (P1-P4) were devised for RNA purification(Table 3) and compared with regards to RNA recovery/yield and purity(FIGS. 6A-6G).

TABLE 3 Process stream Options: Purification → Buffer Exchange 1 TFF(puri b.) → TFF (formulation b.) 2 TFF (no salts) → LC (hydroxyhapatite)→ TFF (formulation b.) 3 GE Core beads (250 KCl) → TFF (formulationb.)/SEC 4 GE Core beads (no → LC (hydroxyhapatite) → TFF (formulationb.) salts)

An in vitro reaction containing a 10-kb RNA replicon of interest wasused as the starting sample.

RNA purity was related to the level of protein (T7 polymerase, cappingenzyme, RNase inhibitor, pyrophosphatase, E. coli proteins carried overfrom DNA template amplification), plasmid DNA and nucleotide after eachstep. RNA recovery and nucleotide levels were measured using the methodsof Example 1. Protein levels were measured using ELISA or polyacryl amidgel electrophoresis followed by silver staining or antibody-baseddetection (western blot). DNA levels were measured by quantitative PCR.

A step of tangential flow filtration can be used to exchange buffer butwhere this results in increased purity it is also a purification step.

For purposes of comparison, a step of DNA digestion using DNase wasperformed for all processes after IVT and before applying the sample tothe chromatography/filtration system. However, it should be noted thatthis step is not mandatory for example where hydroxyapatitechromatography is used.

FIGS. 7A-7G show that protein carryover (T7 and capping enzyme) isobserved only with process 1. Hydroxyapatite chromatography and corebead chromatography can remove protein carry over efficiently. Tracesare detected after purification, below the level of detection of theELISA assay. Core bead flow-through purification followed by tangentialflow filtration is easier to operate that hydroxyapatite chromatography.RNA yield was: P1: 74.8%, P2: 37.3%, P3: 76.2%, P4: 60.7% (RNA recoveryis sumamrized in Table 4). Nucleotide removal was complete in allprocesses in the final product. Process time ranges from 45 mins to 84mins for all processes. DNA concentration in the final product was 0.6ng DNA per 75 μg RNA. The level of E. coli protein contamination wasbelow the detection level of the western blot method used. The apparentRNA particle size as measured by dynamic light scattering in the finalproduct was 40-45 nm radius for all processes.

TABLE 4 Step Overall Step: recovery recovery P1 1 - TFF250 13.8 2 -TFFfb 543.4 74.8 P2 1 - TFF0 81.9 2 - HTP0 77.0 63.0 3 - TFFfb 59.2 37.3P3 1 - CC250 88.7 2 - TFFfb 85.9 76.2 P4 1 - CC0 90.1 2 - HTP0 88.8 79.93 - TFFfb 75.9 60.7

Example 9 Large-Scale Purification of RNA

A combination of tangential flow filtration followed by hydroxyapatitechromatography was used for preparative RNA purification from an invitro transcription reaction sample. An unpurified in vitrotranscription reaction containing 6 mg of a 10-kb RNA capped repliconproduct was used as the starting sample. Tangential flow filtration wasperformed using 10 mM Tris pH 8.0. The RNA-containing fraction wasretained. Potassium chloride was added to the sample at a finalconcentration of 500 mM, and the sample was applied to thehydroxyapatite column (CHT™ Ceramic Hydroxyapatite Type II, 40 μmparticle size, Biorad, in a GE Hi Scale 26 column, 20 cm height, 100 ml;run on a GE ÄKTA explorer 100; flow 10 ml/min; linear velocity 300cm/h). Elution buffers were buffer A (10 mM potassium phosphate, pH 6.5)and buffer B (1M potassium phosphate, pH 6.5). RNA was selectivelyeluted with 18% buffer B (180 mM potassium phosphate). The resultsdemonstrate that this method achieves large-scale, preparative RNApurification with high yield and purity.

A combination of core bead flow-through chromatography followed by TFFwas used for preparative RNA purification from an in vitro transcriptionreaction sample. An unpurified in vitro transcription reactioncontaining 120 mg of a 10-kb capped RNA replicon product was used as thestarting sample. The sample was diluted 4-fold, then potassium chlorideto 250 or 500 mM was optionally added, and the sample was applied to acore bead flow-through column using Capto™Core 700 beads. Chromatographywas performed at a linear flow rate of 275 cm/h (volumetric 25 ml/min)with a contact time of 2.21′. The RNA-containing flow-through was thenfurther purified, concentrated 2-fold, and buffer-exchanged into finalformulation buffer (all in one procedure) using TFF (hollow-fibremodule, 500 kDa cut-off, mPES).

The process was tested with 100 ml capped IVT RNA (about 120 mg), usinga 50 ml Captocore column (Captocore 700, 2.6 cm internal diameter, 10 cmheight run at the conditions described above, flow 25 ml/min) and a 790cm² TFF cartridge (same conditions, flow 200 ml/min). The final materialhad comparable characteristics to the smaller scale process in terms ofactivity, purity and yield. Even in preliminary experiments the processhad a yield of about 80% per step, giving a recovery of 65% overall, andwas completed in 70′ (12 minutes for the Captocore step, 58 minutes forTFF).

The following table shows suitable process parameters for four availablecolumns which can cope with sample volumes of from 10 to 1000 ml:

sample Linear Contact Volume velocity time Internal Area Height Columnvolume Sample/ Dilution final Flow Process (ml) (cm/h) (min) diameter(cm) (cm2) (cm) (ml) CV volume (ml) (ml/min) time (min) GE HiScreen 10275 2.21 0.77 0.47 10.13 4.71 2.12 40 2.1 19 GE HiScale 26/20 100 2752.21 2.60 5.31 10.13 53.75 1.86 400 24.3 16 GE HiScale 26/20 200 2752.21 2.60 5.31 10.13 53.75 3.72 800 24.3 33 Spectra/Chrom 1000 275 2.215.00 19.63 10.13 198.78 5.03 4000 89.9 44 50/100

The table shows flow rate as a linear velocity, which means that thecolumns' internal diameters are irrelevant in defining the method.Linear velocity can be maintained constant in the scaled-up processes.The different column diameter is used to calculate the flow rate inml/min, so as to keep the linear velocity constant and thus to maintainthe same contact time (i.e. the time that the sample stays in thecolumn).

REFERENCES

-   Andrews-Pfannkoch et al. Appl Environ Microbiol. 2010;    76(15):5039-5045.-   Beland et al. J Chromatogr. 1979; 174(1):177-186.-   Bernardi. Nature. 1965; 206:779-783.-   Eon-Duval et al. Anal Biochem. 2003; 316(1):66-73.-   Gennaro, 2000, Remington: The Science and Practice of Pharmacy. 20th    edition, ISBN: 0683306472-   Guerrero-German et al. Bioprocess Biosyst Eng. 2009; 32(5):615-623.-   Kahn et al. Biotechnol Bioeng. 2000; 69(1):101-106.-   Kamalay et al. Proc Natl Acad Sci USA. 1984; 81(9):2801-2805.-   Kendall et al. Biotechnol Bioeng. 2002; 79(7):816-822.-   Løvdal et al. Dis Aquat Organ. 2002; 49(2):123-128.-   Pascolo S. Methods Mol Med. 2006; 127:23-40.-   Zhang et al. PLOS Biol. 2006; 4(1):108-118.

1. A method for purifying RNA from a sample, comprising one or moresteps of tangential flow filtration, hydroxyapatite chromatography, corebead flow-through chromatography, or any combinations thereof.
 2. Themethod according to claim 1, comprising: (a) a step of tangential flowfiltration or core bead flow-through chromatography; and (b) a step ofhydroxyapatite chromatography.
 3. The method according to claim 1,wherein the RNA is purified on a preparative scale.
 4. The methodaccording to claim 1, wherein the method does not comprise the use oflithium chloride, organic solvents, temperatures greater than 70° C.,and/or enzymatic digestion of DNA.
 5. The method according to claim 1,wherein the method includes discarding materials which do not containRNA or the desired RNA species, and maintaining materials which docontain RNA or the desired RNA.
 6. The method according to claim 1,wherein the sample contains RNA and one or more of: plasmid DNA,deoxy-oligonucleotides, deoxynucleoside monophosphates, ribonucleosidetriphosphates and protein; and optionally wherein the sample does notcontain genomic DNA and/or a cell membrane or fragments thereof, forexample wherein the sample is an in vitro transcription reaction sample.7. The method according to claim 1, wherein the sample contains RNA andnot more than four of: plasmid DNA, deoxy-oligonucleotides,deoxynucleoside monophosphates, ribonucleoside triphosphates andprotein; and optionally wherein the sample does not contain genomic DNAand/or a cell membrane or fragments thereof.
 8. The method according toclaim 1, wherein the RNA is a single-stranded RNA, for example an mRNA.9. The method according to claim 1, wherein the RNA comprises a linearsequence of at least 1,000 nucleotides e.g. at least 5,000 nucleotides.10. The method according to claim 1, wherein the method uses ahydrophilic stationary phase and/or a hydrophilic membrane.
 11. Themethod according to any claim 1, wherein the method further comprisesone or more steps of buffer exchange comprising tangential flowfiltration.
 12. The method according to claim 1, wherein the methodfurther comprises a step of RNA manufacture, for example using in vitrotranscription of RNA.
 13. A method for purifying RNA from a sample,wherein the RNA is purified to at least 99% purity in less than 12hours.
 14. A method for purifying RNA from a sample which contains RNA,DNA, pyrophosphates, and free nucleotides, wherein the method providesfinal material in less than 12 hours which is free from DNA,pyrophosphates, and free nucleotides.
 15. A method for preparing apharmaceutical composition comprising the steps of: (a) purifying RNAaccording to a method according to claim 1; and (b) formulating thepurified RNA as a pharmaceutical composition.
 16. A pharmaceuticalcomposition prepared by a method according of claim 15, wherein thepharmaceutical composition is substantially free from one or more of thefollowing: DNA, deoxy-oligonucleotides, deoxynucleoside monophosphates,ribonucleoside triphosphates, polymerase enzyme and RNA capping enzyme.17. The pharmaceutical composition according to claim 16, for use inmedicine.